Wnts (wingless and int-related proteins) are a family of secreted cysteine-rich glycoproteins, expressed in a variety of tissues in developing embryos, thought to be involved in cell fate specification and stem cell commitment. To identify the specific Wnts involved in osteoblastic differentiation of human mesenchymal stem cells (hMSCs), we performed degenerative RT-PCR cloning method to amplify Wnt-encoding cDNAs expressed during osteoblastic differentiation of hMSCs in vitro and during hMSC-directed ectopic osteogenesis in the severe combined immunodeficient (SCID) mouse host. WNT5A was found to be the dominant Wnt expressed during osteoblastic differentiation of hMSCs both in vitro and in vivo. RT-PCR further revealed that hWNT5A and its receptor Frizzled family member 5 (hFZD5) was up-regulated during osteoblastic differentiation compared to uncommitted hMSCs. To evaluate the function of Wnt5a, calvarial cells were obtained from Wnt5a(-/-), Wnt5a(+/-), and wild type mice. Wnt5a(-/-) cells showed significantly slower growth when compared to Wnt5a(+/-) and wild type cells. Gene expression profiles of the Wnt5a(-/-) calvarial cells as compared to wild type cells were evaluated using microarray analysis. 255 genes exhibited at least 2-fold changes in expression. Clusters of genes regulating cell cycle, cell proliferation and cell growth, and gene transcription were altered with absence of Wnt5a expression. In addition, genes regulating osteoblastic differentiation including Runx2, osterix, and alkaline phosphatase (ALP) were shown to be down-regulated in Wnt5a(-/-) cells. In conclusion, Wnt5a is highly expressed during osteoblastic differentiation. Its function during mesenchymal stem cell differentiation as well as cell growth was suggested by comparing the gene expression profile of calvarial cells from the Wnt5a(-/-) and wild type mice.