"It was deemed that Andrew's ankle did not demonstrate sufficient improvement after his first bowling session today (Wednesday)," said an ECB statement, "and in order to allow the ankle to rehabilitate effectively and continue to improve it was felt the best decision was to rule him out now without a further assessment tomorrow (Thursday)."
The statement added: "Andrew will leave the England squad tonight (Wednesday) and return to Lancashire where he will continue to undergo rehabilitation and revue.
"He will only begin to bowl at an appropriate stage and any participation in Lancashire's forthcoming county matches will be made in due course."
The second Test of a four-match series is due to start at Headingley on Friday.
Pace bowling all-rounder Flintoff missed the drawn series opener completed at Lord's on Monday after suffering discomfort on his left ankle, operated on twice over the last three years, during Lancashire's drawn championship match with Hampshire earlier this month.
His unavailability means England are unlikely to drop either all-rounder Paul Collingwood or Ian Bell, two of their four first innings centurions at Lord's, which might have been the case had fit-again captain Michael Vaughan and Flintoff both returned to the team at Headingley.
Now it looks as if Vaughan will replace Owais Shah.
Fast bowler Matthew Hoggard had already been ruled out of the second Test after leaving the field at Lord's in his 11th over with a thigh injury that kept him out of action for the remainder of the game.
Meanwhile Ryan Sidebottom, James Anderson and Liam Plunkett are set to compete for the two remaining places in what is likely to be a four-man attack featuring express quick Stephen Harmison and left-arm spinner Monty Panesar.