Heat stress (HS) suppresses cell proliferation by activating cell cycle checkpoints. Although Wee1 and Chk1/2 regulate S-phase progression and mitotic entry, the role of Wee1 under HS remains unclear. In this study, we investigated whether inhibition of Wee1 and Chk1/2 enhances HS-induced cell death by disrupting cell cycle delay or arrest. HeLa S3 cells were treated with the Wee1 inhibitor MK-1775 and/or the Chk1/2 inhibitor AZD-7762 and then exposed to HS (44°C, 45 min). DNA synthesis, cell cycle progression, protein expression, and cell survival were analyzed by EdU incorporation assay, flow cytometry, immunoblotting, MTT, and clonogenic assay. HS transiently suppressed DNA synthesis and induced G2/M accumulation. MK-1775 or AZD-7762 accelerated recovery of EdU incorporation, with the greatest effect observed after combined treatment. Dual inhibition also increased γH2AX expression, indicating cell cycle progression despite persistent DNA damage or replication stress. Furthermore, combined inhibition promoted mitotic entry, increased the SubG1 population, and markedly reduced cell viability and clonogenic survival. These results indicate that Wee1 and Chk1/2 cooperatively regulate S-phase progression and G2/M checkpoint activation following HS, thereby promoting cell survival. Their simultaneous inhibition drives premature cell cycle progression and enhances hyperthermia-induced cell death, suggesting a promising strategy to improve the therapeutic efficacy of hyperthermia.