England had hoped their star all-rounder would be fit enough to captain the team in the second Test against Pakistan which starts at his Old Trafford home ground on Thursday.
But, after a specialist examined Flintoff's problem left ankle Saturday, England decided the hero of last year's Ashes series would have to undergo a new bout of surgery which will sideline him for at least 12 weeks.
Flintoff now joins fellow Ashes-winners Michael Vaughan (knee), left-arm spinner Ashley Giles (hip) and fast bowler Simon Jones (knee) on the sidelines.
Not only will the 28-year-old Flintoff, who missed the 5-0 one-day series thrashing by Sri Lanka and the drawn first Test against Pakistan, be absent from the rest of the Pakistan Test campaign and subsequent one-dayers, but his participation in October's Champions Trophy event in India is also in doubt.
However, even though the first Test against Australia starts at Brisbane's Gabba ground on November 23, England remain confident that the man they want to lead their Ashes defence in the absence of Vaughan will be fit by then.
After two Twenty20 games for Lancashire, Flintoff had looked on course for a return from an injury which had its origins in floating bone fragments, left over from an operation on the same ankle at the beginning of last year.
But fresh discomfort, suffered on the final day of Lancashire's County Championship defeat away to Kent on Friday, led to an appointment with the specialist and the news every England fan dreaded.
The fact that Flintoff, just like Vaughan, has broken down on his return from injury, led former captain Michael Atherton to question England's medical staff, saying the current stars' fitness struggles were part of what had been "a regular problem for England players" in recent years.
Meanwhile, England's chairman of selectors David Graveney said of Flintoff: "You obviously feel really sorry for him.
"Like Vaughan he has worked really hard for the last four of five weeks to get to this point.
"I don't think I've ever seen him as fit as he looks at the moment, apart from his ankle, but if the predictions for the rehabilitation and recovery are on course he will be fit before we set off for Australia.
"It's a desperately disappointing time for him and his family and your thoughts go out to them."
Justin Langer, one of the Australia players who witnessed first-hand many of Flintoff's greatest moments while the paceman and hard-hitting batsman took 24 wickets and 402 runs during England's 2-1 home Ashes series win last year, was in no doubt about the significance of this latest setback.
"You never like to see a fellow cricketer injured," Langer, in England to play county cricket for Somerset, told Sky Sports. "But from an opening batsman's point of view, I'm delighted."
Langer's former Middlesex team-mate Andrew Strauss, who led England in the first Test at Lord's, is now set to be appointed captain for the remainder of the home season as his side try to come to terms with a mounting injury crisis.
Graveney added: "We are having a bad run of luck with injuries but you have to remember Pakistan are also missing a few players through injury.
"Every player in a team is important but some are more important than others because it affects the balance of the whole line-up," he added.
"Losing your captain, key all-rounder and senior spinner all in the same summer is hard to bare, but we just have to get on with it."
Ian Bell, who had looked as if he would make way for Flintoff despite an unbeaten hundred at Lord's, is now set to retain his place.
That match saw England field a four-man, rather than five-man, attack and, in the talisman's absence they never looked like bowling Pakistan out twice.
And that problem is one that remains for the selectors as they contemplate their Old Trafford line-up.
Meanwhile, Pakistan are still without the injured pace trio of Mohammad Asif, Rana Naved-ul-Hasan and Shoaib Akhtar.
Some reports have suggested that Akhtar, Pakistan's quickest bowler, could be back as soon as next month's third Test at Headingley.
But Pakistan coach Bob Woolmer was far from certain Akhtar would recover from his stress fracture of the ankle even by the time of the fourth and final Test starting at The Oval on August 17.
Woolmer told The News, Pakistan: "It is going to take time ... I would consider Shoaib lucky if he plays in the last Test."