Danny Rose and Alex McCarthy have withdrawn from the England squad for the matches against Croatia and Spain through injury.
Both players arrived at the England camp with injuries, and have now returned to their clubs.
As a result, Gareth Southgate will be without the pair for the Nations League matches against Croatia and Spain, live on Sky Sports.
It remains to be seen whether the manager will call up replacements for either Rose or McCarthy.
Rose was the most senior left wing-back in Southgate's squad. However, following Luke Shaw's earlier withdrawal, Leicester City defender Ben Chilwell now looks set to start in that role, in what would be just his second cap.
McCarthy's withdrawal is less problematic for Southgate. The Southampton goalkeeper is yet to play for England, and Marcus Bettinelli, Jack Butland and Jordan Pickford are already in the squad.
Danny Welbeck is another player who arrived for the Nations League matches with a knock, but he currently remains with the squad.
The Arsenal forward picked up the problem in the Gunners' 5-1 win at Fulham at the weekend, and is unlikely to play against Croatia on Friday.
However, he will travel to the match in Rijeka - which will be played behind closed doors - and the FA are hopeful he will be fit to play against Spain in Seville on Monday.