England had hoped that their talismanic all-rounder would be fit enough to lead them at his Old Trafford home ground, where the second match of the four-Test series against Pakistan starts on Thursday.
He has missed the 5-0 one-day series whitewash against Sri Lanka and last week's drawn first Test agianst Pakistan at Lord's and a return was always conditional on him coming through Lancashire's County Championship clash away to Kent at Canterbury.
For the first three days of that match everything was on course but on Friday's fourth and final day Flintoff suffered fresh discomfort in his ankle after bowling two overs.
If the specialist decides that four weeks rest has not done the trick then surgery would appear to be the only other option - and that would sideline Flintoff for the rest of the English season.
A statement issued by the England and Wales Cricket Board (ECB) said: "Andrew Flintoff has made good progress with his rehabilitation from an injury to his left ankle. He has worked extremely hard and at every stage of the rehabilitation program has shown improvement.
"However, bowling in a four-day county championship match against Kent was intended as the final test of the progress that he has made.
"Unfortunately, he has experienced some discomfort today (Friday) and he will see a specialist tomorrow (Saturday) to reassess the ankle and future management of the injury.
"A decision as to whether he is available for the second Test against Pakistan will be made following his visit to the specialist tomorrow."
Flintoff bowled 19 overs in Kent's first innings, taking two for 45, but only sent down four in all second time around.
He first felt discomfort in his left ankle during the final Test against Sri Lanka at Trent Bridge nearly two months ago.
A scan revealed that bone fragments, left over from an operation on the same ankle at the beginning of last year, could be causing the discomfort.
England had just four frontline bowlers on show at Lord's and, without Flintoff, they lacked the firepower to bowl Pakistan out twice on a good pitch.
If Flintoff doesn't make it, Warwickshire batsman Ian Bell, who was set to be axed despite scoring an unbeaten hundred at Lord's, is set to be reprieved.
And were England to field a similar line-up to last week's, Bell and Paul Collingwood would provide medium-pace support to senior quicks Stephen Harmison and Matthew Hoggard, with Liam Plunkett, Jon Lewis and Sajid Mahmood battling for the third seamer role.
The Old Trafford pitch traditionally offers assistance to spinners but, in Flintoff's absence, left-armer Monty Panesar is likely to be England's lone specialist slow bowler.
However, either one of off-spin duo Alex Loudon and Jamie Dalrymple could be in contention if England do decide they need a second spinner.
Andrew Strauss, England's captain at Lord's, is set to lead the side again if Flintoff is unavailable.