Ror2 is a receptor tyrosine kinase whose expression increases during differentiation of pluripotent stem cells to osteoblasts and then declines as cells progress to osteocytes. To test if Ror2 plays a role in osteoblastogenesis, we investigated the effects of Ror2 over-expression and down-regulation on osteoblastic lineage commitment and differentiation. Expression of Ror2 in pluripotent human mesenchymal stem cells (hMSC) by adenoviral infection caused formation of mineralized extracellular matrix, which is the ultimate phenotype of an osteogenic tissue. Concomitantly, Ror2 over-expression inhibited adipogenic differentiation of hMSC as monitored by lipid formation. Ror2 shifted hMSC fate toward osteoblastogenesis by inducing osteogenic transcription factor osterix, and suppressing adipogenic transcription factors CCAAT/enhancer-binding protein and peroxisome proliferator activated receptor . Infection with Ror2 virus also strongly promoted matrix mineralization in committed osteoblast-like MC3T3-E1 cells. Expression of Ror2 in a human pre-osteocytic cell line by stable transfection also promoted further differentiation as judged by inhibited alkaline phosphatase activity, potentiated osteocalcin secretion and increased cellular apoptosis. In contrast, down-regulation of Ror2 expression by shRNA essentially abrogated dexamethasone-induced mineralization of hMSC. Furthermore, down-regulation of Ror2 expression in fully differentiated SaOS-2 osteosarcoma cells inhibited alkaline phosphatase activity. We conclude that Ror2 initiates commitment of MSC to osteoblastic lineage and promotes differentiation at early and late stages of osteoblastogenesis. Finally, utilizing a mouse calvariae ex vivo organ culture model, we demonstrate that these effects of Ror2 result in increased bone formation, suggesting that it may also activate mature osteoblasts.