Tammy Abraham, Ben Chilwell and Jadon Sancho will miss England’s game against Wales on Thursday.
The FA is continuing its investigation into the players’ attendance at a party on Saturday night, which broke the government’s coronavirus rules.
Abraham’s friends and family threw a surprise party to celebrate his 23rd birthday at his London flat, with Chilwell and Sancho also there.
Around 20 people in total were inside the flat, in a clear breach of the national ‘rule of six’.
The FA told all three to stay away from St George’s Park on Monday, while the rest of the England players signed in and had their regular coronavirus test.
The whole squad will be tested again on Tuesday, two days before the Wales game, in line with UEFA’s protocols.
But there are no plans for Sancho, Chilwell and Abraham to be re-admitted into the squad by then, and so – having missed the compulsory tests – there is no way they can be included in Southgate’s team to play in the all-British friendly at Wembley.
In a statement on Monday, Abraham said: “On Saturday evening, I arrived home to find a small surprise gathering had been organised for me, with family and close friends, to celebrate my birthday.
“Although I was totally unaware this was planned, I would like to wholeheartedly apologise for the naivety shown by all for the organisation and attending of this gathering.
“I recognise that I have a responsibility both in my professional and personal life to honour and respect the guidelines and deeply regret that this took place. All I can do now is learn from it, apologise to everyone and ensure it never happens again.”
Chilwell and Sancho also both apologised for attending the party and pledged to “learn from this”.
Southgate gave the squad specific warnings about breaching the rules after the embarrassing situation in Reykjavik four weeks ago, when Phil Foden and Mason Greenwood were sent home for leaving the eco-bubble to meet two Icelandic women.
Southgate has yet to decide if Sancho, Chilwell and Abraham will be allowed back into the England camp in time for the two competitive Nations League games against Belgium and Denmark.
It is feasible for them to join up with the squad, if FA officials arrange for them to be tested on Friday – two days before the Belgium game at Wembley on Sunday.