Osterix is a bone-related transcription factor that functions genetically downstream of Runx2, which controls both growth and differentiation in osteoblasts. Here we assessed the biological function of Osterix in mesenchymal cells that are not normally committed to the osteogenic lineage. Stably transfected NIH3T3 fibroblasts that express exogenous Osterix were examined for their ability to convert into osteoblastic cells by analyzing gene expression profiles of bone phenotype related markers, as well as by measuring bone nodule formation and cell proliferation. Forced expression of Osterix stimulates osteopontin gene expression but not the expression or activity of other bone-related markers, including collagen type I, alkaline phosphatase, osteocalcin, or osteonectin. Moreover, cells stably expressing Osterix do not induce bone nodule formation. Strikingly, both polyclonal and monoclonal cells expressing Osterix exhibit enhanced proliferation. Collectively, these results indicate that Osterix is insufficient to establish osteogenic lineage commitment, perhaps due to the ability of Osterix to promote cell growth. We propose that regulatory pathways operating upstream of or in parallel with Osterix are required for osteogenic conversion of uncommitted mesenchymal cells.