Oxygen availability is a critical signal for proper development of many tissues, however there is limited knowledge of its role in the maturation of bone cells. To test the hypothesis that low pO₂ regulates bone cell mineralization, MLO-A5 and MLO-Y4 cells were cultured in monolayer and three-dimensional alginate scaffolds in hypoxia (2% O₂) or normoxia (20% O₂). Hypoxia reduced mineralization and decreased alkaline phosphatase activity of preosteocyte-like MLO-A5 cells in both monolayer and alginate cultures. Similar changes in osteogenic activity were seen when the were subjected to chemical hypoxia. Likewise, Osteocyte-like MLO-Y4 cells also exhibited reduced osteogenic activity in hypoxia relative to normoxic controls. Based on these observations, it is concluded that a low pO₂ decreased the mineralization potential of bone cells at both early and late stages of maturation. Since the oxemic state is transduced by the transcription factor, HIF-1α, experiments were performed to determine if this protein was responsible for the observed changes in mineral formation. It was noted that when HIF-1α was silenced, mineralization activities were not restored. Indeed, in hypoxia, in relationship to wild type controls, the mineralization potential of the knockdown cells was further reduced. Based on these findings, it is concluded that the osteogenic activity of preosteocyte-like cells is dependent on both the O₂ tension and the expression of HIF-1α.