Dominic Calvert-Lewin, Bukayo Saka and Harvey Barnes have been handed their first senior England call-ups, while Mason Greenwood and Phil Foden have been dropped.
After hitting a career-best 15 goals last term, Calvert-Lewin's impressive start to the new campaign has propelled Everton to three wins from three in the Premier League.
The 23-year-old is the Premier League's joint-leading goalscorer with five, and also scored a hat-trick for the Toffees in Wednesday's Carabao Cup win over West Ham.
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Saka, 19, has featured just three times for Arsenal during this campaign but broke into the first-team and impressed while playing at full-back for the Gunners last season.
Barnes meanwhile has helped Leicester to the top of the early Premier League table, with the 22-year-old scoring in the Foxes' win over Burnley.
As expected, Southgate has left out Greenwood and Foden after the pair were sent home from Iceland last month for breaching coronavirus protocols by meeting up with two women inside the public areas of the team hotel.
Manchester United captain Harry Maguire has been recalled, after he was dropped from the last squad following his convictions for assault and attempted bribery in a Greek court, which he has since appealed with the convictions nullified ahead of a full retrial.
Having handed first-time call-ups to six players for last month's matches, Southgate has a more experienced squad this time, with fit-again Ben Chilwell, Harry Winks and Marcus Rashford returning to the fold.
Liverpool captain Jordan Henderson is also named in the squad despite picking up an injury issue in the Reds' win over Chelsea.
England have three home games during this international break, including Nations League games against Belgium and Denmark and a friendly against Wales.
England squad in full
Goalkeepers: Dean Henderson (Manchester United), Nick Pope (Burnley), Jordan Pickford (Everton).
Defenders: Trent Alexander-Arnold (Liverpool), Ben Chilwell (Chelsea), Conor Coady (Wolves), Eric Dier (Tottenham), Joe Gomez (Liverpool), Michael Keane (Everton), Harry Maguire (Manchester United), Ainsley Maitland-Niles (Arsenal), Tyrone Mings (Aston Villa), Bukayo Saka (Arsenal), Kieran Trippier (Atletico Madrid), Kyle Walker (Manchester City).
Midfielders: Jordan Henderson (Liverpool), Mason Mount (Chelsea), Kalvin Phillips (Leeds United), Declan Rice (West Ham), James Ward-Prowse (Southampton), Harry Winks (Tottenham).
Forwards: Tammy Abraham (Chelsea), Harvey Barnes (Leicester), Dominic Calvert-Lewin (Everton), Jack Grealish (Aston Villa), Danny Ings (Southampton), Harry Kane (Tottenham), Marcus Rashford (Manchester United), Jadon Sancho (Borussia Dortmund), Raheem Sterling (Manchester City).
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'Foden, Greenwood have suffered enough'
Southgate says the decision to exclude Foden and Greenwood was important in order to "send a message to all our players".
However, the England boss revealed he will not punish the pair further and hopes their omission will allow time to reflect.
"They're two very talented players," Southgate said. "We have to send a message to all our players, at every age level, that that sort of thing isn't how we do things at England.
"But, we've also got to rehabilitate these two boys. Young people will make mistakes.
"These boys have suffered enough. We think they needed this period of reflection, but then that gives them the opportunity to get back on their feet with their clubs, enjoy their football again and then I can speak to them after."
Southgate: Every club deserves our respect
Southgate has revealed he plans to rotate heavily between England's fixtures and says no player in his 30-man squad is likely to start in all three matches.
A number of Premier League managers, including Tottenham's Jose Mourinho, have expressed concerns over their players' fitness levels.
Speaking ahead of their Europa League qualifier against Maccabi Haifa on Thursday, Mourinho said he hopes England's coaches "respect" his Tottenham players, but insisted he would not "call or ask or beg."
Asked about Mourinho's comments, Southgate joked: "He won't phone, but he will stand on top of Tottenham's stadium and tell us."
He added: "We're looking at pretty much all the players getting game time, ideally. We want to learn things from these matches.
"We're certainly not looking to start players in all three games. I can't guarantee that no player will play no minute of each game.
"The life of an England manager is very complicated. Every club deserves our respect, whatever position they are in the league.
"Of course, the workload for the clubs at the top is higher, but those playing further down are no less important.
"We've got to work together where we can. Where we can help we will but equally we've got to win games of football and develop as a team.
"The 30-man squad is very unusual for us. That's a nod to the fact we've got three matches, but also the amount of football the players are currently experiencing. There's a lot of physical issues that all clubs and coaches are having to deal with."
Sancho misses Dortmund training despite England selection
Sancho was not present at Borussia Dortmund's training session on Thursday due to an ongoing illness, despite receiving a call up for Southgate's squad the same afternoon.
Sancho did not drive into the Brackel training ground in North Rhine-Westphalia, as members of Lucien Favre's squad began to arrive for coronavirus testing from 12pm and for afternoon practice, which began at 3pm.
Sky Sports News has been told he is still suffering from the "respiratory infection" which prevented him from travelling with the team for Wednesday's German Supercup final against Bayern Munich.
This in turn will mean he is unlikely to play against Freiburg in the Bundesliga on Saturday, and it remains unclear whether he has been able to take a COVID-19 test at home or elsewhere.
England's players are due to report for international duty at St George's Park on October 5, which will coincide with the transfer deadline day.
Borussia Dortmund continue to emphasise that Sancho will not be sold this window, while Manchester United are expected to table another bid for him this week, after their £91.3m (€100m) offer was rejected on Monday.
Speaking after Everton's win on Wednesday, Calvert-Lewin said it would be a "dream come true" to represent England.
He has benefitted from being the focal point in an attacking trident involving Richarlison and James Rodriguez - and the England manager will be encouraged by Ancelotti's tactical alteration to a 4-3-3 system that can similarly be deployed by the national team.
Calvert-Lewin became the first player to score in each of Everton's first three Premier League games in a single campaign since Steven Naismith in 2014/15 - and with the Wales friendly and Denmark encounter straddling the Belgium showdown, Southgate has the chance to try out a functioning Plan B in the event of Harry Kane's absence.
Speaking on the Kings of the Premier League show, which will return to screens this Friday on Sky Sports Premier League at 8pm, Jamie Carragher said: "There isn't much competition with Harry Kane for the actual centre-forward spot. There are a lot of wide forwards, when you think of Marcus Rashford, Raheem Sterling and Jadon Sancho.
"But, in terms of real strikers, now Jamie Vardy has retired from international duty, you look at Danny Ings, maybe Tammy Abraham - they've had sniffs with England before, but Calvert-Lewin is as good as those two.
"He's been outstanding and had a great start. Carlo Ancelotti has had some of the best strikers in world football at times. He's put his faith in Calvert-Lewin and it's paying off. I'm a big fan."
Watch England in the Nations League on Sky Sports
Watch England's Nations League games against Belgium (Oct 11) and Denmark (Oct 14) live on Sky Sports Main Event.
From England's quest to improve on their third-place finish in last year's competition to Scotland, Wales and Northern Ireland's pursuit of promotion from League B, we will bring you all the twists and turns along the way.
As well as our live coverage, if you are out and about, you can follow all of those Home Nations games on the Sky Sports website and app, where we will have minute-by-minute updates and show you the goals as they go in.
We will also have highlights of every game involving the Home Nations and the best of the other Nations League fixtures available too.