Abstract: Heat shock (HS)–induced stress response in human cells results in a variety of biological effects and is known to induce the transcription of heat-shock proteins, which help the cells to cope with different kinds of stress. We have studied the effects of HS on the differentiation of human mesenchymal stem cells (hMSCs) into osteoblastic cells. As a model for hMSCs we used a telomerase-immortalized hMSC line designated hMSC-TERT. Cells were exposed to 1 h HS at 41°C, 42.5°C, or 44°C prior to incubation in a medium containing either 10−8 M 1α,25-dihydroxy-vitamin-D3 (calcitriol) or 10−8 M calcitriol, 50 μg/mL l-ascorbic acid, and 10 mM β-glycerophosphate followed by an analysis of induction of osteoblast differentiation and the formation of mineralized matrix, respectively. Our results indicate that the exposure of cells to mild heat stress enhances the extent of differentiation of hMSCs by 12% to 42%. These effects are an expression of the phenomenon of mild stress-induced hormesis.