Adam Lallana has been ruled out of England’s games against Spain and Switzerland after suffering a groin strain.
The 30-year-old started Tuesday’s training session at St George’s Park with the rest of the 22-man squad but pulled up and left early to be assessed.
England have since confirmed Lallana, who has struggled with injuries over the past year, will return to his club for treatment and take no further part in the first UEFA Nations League matches.
Injury issues cost Lallana a place in the squad that reached the World Cup semi-finals during the summer, after working with the team throughout their preparation camp.
Southgate’s squad is already down to 22 men after Raheem Sterling withdrew on Monday evening with a back problem.
The remaining players trained together for the first time since the World Cup on Tuesday morning, when Liverpool striker Dominic Solanke was brought up from the Under-21s to bolster numbers.
Those involved in Premier League action on Sunday left the session early, including rejuvenated Manchester United left-back Luke Shaw.