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Introduction: Skeletal homeostasis is normally maintained by the stability between bone formation by osteoblasts and bone resorption by osteoclasts. However, the correlation between the inflammatory reaction and osteoblastic differentiation of cultured osteoprogenitor cells has not been fully investigated. This study examined the effects of inflammatory cytokines on the osteoblastic differentiation of cultured human periosteal-derived cells. Materials and Methods: Periosteal-derived cells were obtained from the mandibular periosteum and introduced into the cell culture. After passage 3, the periosteal-derived cells were further cultured in an osteogenic induction Dulbecco’s modified Eagle’s medium (DMEM) medium containing dexamethasone, ascorbic acid, and β-glycerophosphate. In this culture medium, tumor necrosis factor (TNF)-αwith different concentrations (0.1, 1,and 10 ng/mL) or interleukin (IL)-1βwith different concentrations (0.01, 0.1, and 1 ng/mL) were added. Results: Both TNF-αand IL-1βstimulated alkaline phosphatase (ALP) expression in the periosteal-derived cells. TNF-αand IL-1βincreased the level of ALP expression in a dose-dependent manner. Both TNF-αand IL-1βalso increased the level of alizarin red S staining in a dose-dependent manner during osteoblastic differentiation of cultured human periosteal-derived cells. Conclusion: These results suggest that inflammatory cytokines TNF-αand IL-1βcan stimulate the osteoblastic activity of cultured human periosteal-derived cells.