Mammalian cells respond to environmental stress by activating heat shock transcription factors (eg, Hsf1) that regulate increased synthesis of heat shock proteins (Hsps). Hsps prevent the disruption of normal cellular mitosis, meiosis, or differentiation by environmental stressors. To further characterize this stress response, transformed wild-type Hsf1 ^/ and mutant Hsf1^−/− mouse embryonic fibroblasts (MEFs) were exposed to (1) lethal heat (45°C, 60 minutes), (2) conditioning heat (43°C, 30 minutes), or (3) conditioning followed by lethal heat. Western blot analysis demonstrated that only Hsf1 ^/ MEFs expressed inducible Hsp70s and Hsp25 following conditioning or conditioning and lethal heat. Exposure of either Hsf1 ^/ or Hsf1^−/− MEFs to lethal heat resulted in cell death. However, if conditioning heat was applied 6 hours before lethal heat, more than 85% of Hsf1 ^/ MEFs survived, and cells in G₂/M transiently increased 3-fold. In contrast, conditioned Hsf1^−/− MEFs neither survived lethal heat nor exhibited this G₂/M accumulation. Coinfection with adenoviral Hsp70 and Hsp25 constructs did not fully recreate thermotolerance in either Hsf1 ^/ or Hsf1^−/− MEFs, indicating other Hsf1-mediated gene expression is required for complete thermotolerance. These results demonstrate the necessity of Hsf1-mediated gene expression for thermotolerance and the involvement of cell cycle regulation, particularly the G₂/M transition, in this thermotolerant response.