Mesenchymal stem cells (MSCs) are multipotent cells that can replicate and differentiate to different lineages, potentiating their use as integral components in regenerated mesenchymal tissues. Our previous work and other studies have indicated that mild heat shock enhances osteogenesis. However, the influence of pro-inflammatory cytokines on osteogenic differentiation during mildly elevated temperature conditions remains to be fully explored. In this study, human MSCs (hMSCs) were cultured with tumor necrosis factor-alpha (TNF-α), an important mediator of the acute phase response, and interleukin-6 (IL-6) which plays a role in damaging chronic inflammation, then heat shocked at 39 °C in varying frequencies—1 h per week (low), 1 h every other day (mild), and 1 h intervals three times per day every other day (high). DNA data showed that periodic mild heating inhibited suppression of cell growth caused by cytokines and induced maximal proliferation of hMSCs while high heating had the opposite effect. Quantitative osteogenesis assays show significantly higher levels of alkaline phosphatase (ALP) activity and calcium precipitation in osteogenic cultures following mild heating compared to low heating or nonheated controls. These results demonstrate that periodic mild hyperthermia may be used to facilitate bone regeneration using hMSCs, and therefore may influence the design of heat-based therapies in vivo.