England's Kyle Walker, Harry Maguire and Sam Johnstone have all withdrawn from the squad ahead of the Three Lions' friendly with Belgium on Tuesday.
In their place, James Trafford and Rico Lewis have been called up to the senior squad.
Walker and Maguire were set to start in Saturday's match against Brazil, where England suffered their first Wembley defeat since October 2020.
The right back had to be substituted after just 19 minutes due to a hamstring issue, and Gareth Southgate was unsure of the severity of the injury initially.
However, Walker has now withdrawn from the squad, casting doubt on his availability for Manchester City's clash with Arsenal on March 31.
Maguire, who took over the captain's armband from Walker, was substituted in the 67th minute against Brazil.
While no injury was mentioned at the time, he has since developed an unexplained problem that requires assessment.
With Johnstone also absent, Burnley's Trafford has been promoted to the senior squad for the first time.
The 21-year-old, who played every minute for England's Under-21s in their Euro 2023 triumph, started the Premier League season as Burnley's first-choice goalkeeper but has recently lost his place in the team.
Manchester City's Lewis, filling in for the injured Walker, has also been called up.
The 19-year-old earned his first senior cap in November's Euro 2024 qualifying draw with North Macedonia.
Southgate has confirmed that Harry Kane, Cole Palmer and Jordan Henderson will also miss the Belgium clash due to injury. Palmer and Henderson are undergoing late fitness tests.
