Previous studies have shown that subjecting Drosophila melanogaster flies to a mild stress at young or middle age can increase lifespan and resistance to severe stresses throughout life and that the NF-κB-like transcription factor DIF, the 70 kDa heat-shock proteins, and the Drosophila Forkhead box class O (dFOXO) transcription factor could explain some of these effects. The present study showed that two dFOXO mutants do not survive longer heat if previously subjected to a mild cold stress, contrarily to wild-type flies. This cold pretreatment had nearly no effect on dFOXO nuclear translocation in wild-type males. Heat stress strongly increased dFOXO translocation, but this effect was lowered in cold-pretreated males. Because cold-pretreated wild-type males survived longer heat and had nevertheless a lower dFOXO translocation after this heat stress, one can conclude that dFOXO is required to resist heat but that the cold pretreatment makes that other mechanisms partly substitute to dFOXO translocation.