The Three Lions boss initially called up 33 players due to several fitness doubts and with a large chunk involved in the Champions League and Europa League finals last week, but that group has now been trimmed.
Here, the PA news agency takes a look at the seven players to miss out.
Aaron Ramsdale (Sheffield United, age 23, caps 0, goals 0)
Nick Pope's injury opened the door for a new face to be selected as third-choice goalkeeper, but the nod went to another player who suffered relegation this season, Sam Johnstone. Ramsdale, like the West Brom custodian, was a busy man between the sticks and produced some strong displays for Sheffield United, but was edged out by a rival who was with the England camp in March.
Ben White (Brighton, 23, 0, 0)
The 23-year-old was a surprise inclusion in Southgate's bigger group, but was rewarded for an impressive debut season in the top flight. The centre-back had also impressed during Leeds' promotion-winning campaign two years ago with his flexibility viewed as a big asset. While White is adept in a back three or back four, it was always expected this tournament was a little too early.
Ben Godfrey (Everton, 23, 0, 0)
Godfrey's versatility may have made him a tempting choice for the Three Lions boss, who would have been aware of the defender due to his rise through the England age groups. A regular with the Under-21s, he played twice at their European Championship in March and caught the eye across several positions for Everton. His lack of experience compared to his rivals at centre-back would have counted against him.
James Ward-Prowse (Southampton, 26, 6, 1)
The dead-ball specialist has been in and around the England camp since Southgate took over but was not taken to the World Cup three years ago and has missed out again. While the Saints captain scored during the fixtures back in March, he has ultimately paid the price for Jordan Henderson's return to fitness and the emergence of Kalvin Phillips of Leeds.
Jesse Lingard (Manchester United, 28, 27, 4)
A successful loan with West Ham got the playmaker's season back on track and he earned a place in Southgate's most recent squad, but the national team manager did admit the United ace was "lucky" to be involved with injuries elsewhere opening up a space. While Lingard finished the campaign with nine goals, too much competition in the attacking areas has seen him drop out.
Ollie Watkins (Aston Villa, 25, 1, 1)
Perhaps the unluckiest of the seven to miss out with England only taking two centre forwards, Watkins enjoyed a memorable international debut in March when he netted on his debut against San Marino and struck 14 times during his first campaign in the Premier League. But the former Brentford striker has not made the cut with more versatile options in attack selected instead.
Mason Greenwood (Manchester United, 19, 1, 0)
The teenager was the first of the seven to miss out when club side Manchester United revealed on Tuesday morning he had withdrawn from the squad in order to "recover fully from an underlying injury" which restricted his game-time in recent months. Although the 19-year-old only has one senior cap, his ability in front of goal put him in the running and eight goals from his last 14 games further added weight to that argument, but Greenwood's time is not now.