There were hopes Flintoff might return on Friday night, almost five years after his last professional match in England's 2009 Ashes triumph at The Oval.
But after he played for Lancashire's 2nd XI in a Twenty20 semi-final against Leicestershire at Arundel on Thursday, it was decided he will not take part after all against Yorkshire.
The all-rounder took two for 39 in his four overs against the Foxes second string and made a run-a-ball 16 at the top of the order on Thursday, before being bowled sweeping at left-arm spinner James Sykes as the Red Rose fell 30 runs short in pursuit of 186 to win.
Lancashire's coaching staff monitored Flintoff's performance closely, following his only previous match for his Northern League club side St Annes last weekend.
Speaking before confirmation that he will not play on Friday, Lancashire coach Mark Chilton backed his former team-mate's bid to play professionally again.
"He is fiercely determined, an incredibly determined character - and he has set himself a challenge to see if he can get back to playing at the level he used to," Chilton told Sky Sports News.
"Knowing him as I do, I wouldn't put it past him.
"I think he's probably got to a stage where he thinks 'I'm at an age now where, if I don't do it now, the time has probably gone'."
One former England player will still be a notable Twenty20 participant on Friday, however.
Surrey have already confirmed Kevin Pietersen - controversially axed by the England and Wales Cricket Board in the aftermath of last winter's Ashes whitewash - will be back for them, after his Indian Premier League stint, against Essex at The Oval.
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