Jordan Henderson, Jack Grealish, Raheem Sterling, Mason Mount and Luke Shaw have withdrawn from the England squad ahead of Monday's World Cup qualifier in San Marino.
The Football Association also confirmed Conor Gallagher, the Chelsea midfielder currently on loan at Crystal Palace, will join the group in its latest squad update.
Gareth Southgate's side only need to avoid defeat against the minnows in Serravalle to secure their place in next year's tournament in Qatar.
Henderson and Grealish will not be available after returning to their clubs, Liverpool and Manchester City respectively, for further assessment on injuries.
City forward Sterling is unavailable due to a personal matter, while Chelsea midfielder Mount requires dental surgery and Manchester United left-back Shaw has suffered a concussion.
Declan Rice, Marcus Rashford and James Ward-Prowse were also named in Gareth Southgate's squad for this set of international fixtures, but the trio had to withdraw due to injury.
Gallagher, meanwhile, has received his first call-up to the England senior squad ahead of Monday's final World Cup qualifier away to San Marino.
The Chelsea loanee has produced some excellent performances for Crystal Palace this season, scoring four goals and providing two assists in 10 games for Patrick Vieira's side, and could now be rewarded with his first senior cap.
Gallagher had been part of England's Under-21 squad but has now moved up to the senior squad.
A product of Chelsea's academy, Gallagher is in his fourth loan spell away from Stamford Bridge, having previously spent time with Charlton Athletic, Swansea and West Brom.
Born in England, he is also eligible to represent Scotland or the Republic of Ireland due to his heritage.
The 21-year-old has represented England at every level from the Under-17s upwards and has played 13 times for the Under-21s.
He was involved as England Under-21s beat the Czech Republic 3-1 in their latest European qualifier at Turf Moor on Thursday.
Leeds' Sam Greenwood will take his place in Lee Carsley's squad ahead of Tuesday's game in Georgia.
Emile Smith Rowe, who also received a late call-up to the squad having initially been selected for the Under-21s, made his senior debut as a substitute in the win against Albania on Friday.