To obtain sufficient numbers of cells for tissue engineering applications, human bone marrow‐derived mesenchymal stem cells (hBM‐MSC) are commonly cultured as monolayers in incubators containing room air. In this study, we investigated whether three‐dimensional (3D) culture conditions and incubator gas concentrations more similar to those observed in vivo impacted on cell expansion, differentiation capability, or phenotype of hBM‐MSC. We found that 3D culture alone increased the expression of some molecules involved in osteogenic and adipogenic differentiation. In contrast, 3D culture did not induce chondrogenic differentiation, but enhanced the response to the chondrogenic differentiation medium. Changing the oxygen concentration to 6% and the carbon dioxide concentration to 7.5% did not impact on the results of any of our assays, showing that the hyperoxia of room air is not detrimental to hBM‐MSC proliferation, differentiation, or phenotype. J. Cell. Biochem. 112: 684–693, 2011. © 2010 Wiley‐Liss, Inc.