Last season, Chesterfield suffered relegation from League One and the same has effectively occurred at the fourth tier with the club nine points adrift of safety with three matches remaining.
Due to the club's negative goal difference, Chesterfield have accepted their fate and decided to act by parting company with Lester, who had been in charge since September.
The 42-year-old told the club's official website: "I've spoken to Ashley Carson at length and I'm desperately sorry I've not been able to save the club from relegation. Looking ahead, with the proposed plans, I felt it correct for the club to move forward without me as manager.
"I would especially like to thank Dave Allen and Ashley Carson for their amazing support. They could have not done any more.
"I would like to thank the Chesterfield fans for their support since my return to the club. Their support through difficult times was, as always, fantastic."
Lester recorded nine wins and seven draws from his 37 matches in charge of the club.