The transcription factor Osterix (Sp7) is essential for osteoblastogenesis and bone formation in mice. Genome wide association studies have demonstrated that Osterix is associated with bone mineral density in humans; however, the molecular significance of Osterix in human osteoblast differentiation is poorly described. In this study we have characterized the role of Osterix in human mesenchymal progenitor cell (hMSC) differentiation. We first analyzed temporal microarray data of primary hMSC treated with bone morphogenetic protein-6 (BMP6) using clustering to identify genes that are associated with Osterix expression. Osterix clusters with a set of osteoblast-associated extracellular matrix (ECM) genes, including bone sialoprotein (BSP) and a novel set of proteoglycans, osteomodulin (OMD), osteoglycin, and asporin. Maximum expression of these genes is dependent upon both the concentration and duration of BMP6 exposure. Next we overexpressed and repressed Osterix in primary hMSC using retrovirus. The enforced expression of Osterix had relatively minor effects on osteoblastic gene expression independent of exogenous BMP6. However, in the presence of BMP6, Osterix overexpression enhanced expression of the aforementioned ECM genes. Additionally, Osterix overexpression enhanced BMP6 induced osteoblast mineralization, while inhibiting hMSC proliferation. Conversely, Osterix knockdown maintained hMSC in an immature state by decreasing expression of these ECM genes and decreasing mineralization and hMSC proliferation. Overexpression of the Osterix regulated gene OMD with retrovirus promoted mineralization of hMSC. These results suggest that Osterix is necessary, but not sufficient for hMSC osteoblast differentiation. Osterix regulates the expression of a set of ECM proteins which are involved in terminal osteoblast differentiation. J. Cell. Physiol. 227: 2677–2685, 2012. © 2011 Wiley Periodicals, Inc.