Oxygen tension plays an important role in the regulation of cellular processes. During hematopoietic stem cell (HSC) differentiation, HSCs migrate from one stem cell niche to the next, each with a different oxygen tension that determines which signaling pathways are on and off, determining the differentiation stage of the cell. Oxygen tension influences osteoblast differentiation and mineralization. Low oxygen levels inhibit matrix formation and mineralization. We were interested in the regulatory mechanisms that underlie this inhibition and wondered whether a switch in oxygen tension could have varying effects depending on the differentiation phase of the osteoblasts. We performed an oxygen tension switch phase study in which we switched osteoblasts from high to low oxygen tension during their 3 week differentiation and mineralization process. We performed microarray expression profiling on samples collected during this 3 week period and analyzed biochemical and histo-chemical endpoint parameters to determine the effect of a switch in oxygen levels on mineralization. We found that low oxygen tension has the most profound impact on mineralization when administered during the period of matrix maturation. Additionally, a large set of genes was regulated by oxygen, independent of the differentiation phase. These genes were involved in cell metabolisms and matrix formation. Our study demonstrates that variation in oxygen tension strongly affects gene expression in differentiating osteoblasts. The magnitude of this change for either expression levels or the number of regulated probes, depends on the osteoblast differentiation stage, with the phase prior to the onset of mineralization being most sensitive. J. Cell. Physiol. 228: 18631872, 2013. (c) 2013 Wiley Periodicals, Inc.