Eric Dier will miss England’s Nations League match with Denmark on Wednesday because of a hamstring injury.
The 26-year-old, who is comfortable in defence or midfield, played in Sunday’s win over world number one side Belgium that put England top of Group B.
Harry Winks, Michael Keane, Harvey Barnes and Tammy Abraham have also been left out of the 23-man squad, published by Uefa.
Ben Chilwell and Kieran Trippier withdrew from the squad on Tuesday.
A pre-existing injury led to the withdrawal of Chelsea’s Chilwell, while Atletico Madrid’s Trippier also left the camp to deal with a Football Association (FA) hearing for alleged betting breaches.
The FA said Trippier had departed the squad “for personal reasons”.
Chilwell, 23, did not feature in either match, having breached Covid-19 guidelines when he reportedly attended a birthday party for fellow Chelsea player Abraham last weekend.
Southgate said of Trippier’s withdrawal: “There is not a lot I can say about that really. It is not something which is in my control.
“I have got to focus on preparing the team with the players I have got available. We always find solutions. It is an opportunity for somebody else.
“If I lose focus on the job in hand – there are a million distractions, frankly – so this is another distraction, but it is something I have got to plough on through.”
Arsenal pair Bukayo Saka and Ainsley Maitland-Niles are the two most obvious alternatives for the left-back role.
Ton up for Eriksen
Inter Milan midfielder Christian Eriksen is in line to win his 100th cap for Denmark at Wembley, where he played for a season with former club Tottenham whwhile n Spurs’ new stadium was being built.
Eriksen, 28, played more than 300 games in seven seasons with Spurs, before moving to Serie A last January.
“It’s good to be back at Wembley and back in London,” he said. “To reach 100 is impressive, in a weird way, because it’s something you only think about when you retire.
“You want to play the next game, and the next one. When I retire, I’ll look back – but at the moment it’s just about the next one.
“You play for Denmark because you are talented and because you work for it.”
England and Denmark played out a drab goalless draw last month but the Danes picked up an impressive 3-0 win over Iceland on Sunday.
Eriksen added: “We’ve come here with two wins in our last two games so we are confident we can get a result.
“We know England are a good team but we learnt a lot from the first game against them and I hope we will take it to the next step.”