Accumulating evidence indicates that extracellular nucleotides, signaling through purinergic receptors, play a significant role in bone remodeling. Mesenchymal stem cells (MSCs) express functional P2Y receptors whose expression level is regulated during osteoblast or adipocyte differentiation. P2Y₁₃-deficient mice were previously shown to exhibit a decreased bone turnover associated with a reduction in the number of both osteoblasts and osteoclasts on the bone surfaces. We therefore examined whether P2Y₁₃R activation was involved in the osteogenic differentiation of MSC. Our study demonstrated that ADP stimulation of P2Y₁₃R^+/+ (but not P2Y₁₃R^-/-) adherent bone marrow stromal cells (BMSCs) increased significantly the formation of alkaline phosphatase-colony-forming units (CFU-ALP) as well as the expression of osteoblastic markers (osterix, alkaline phosphatase, and collagen I) involved in the maturation of preosteoblasts into osteoblasts. The number of CFU-ALP obtained from P2Y₁₃R^-/- BMSC and the level of osteoblastic gene expression after osteogenic stimulation were strongly reduced compared to those obtained in wild-type cell cultures. In contrast, when P2Y₁₃R^-/- BMSCs were incubated in an adipogenic medium, the number of adipocytes generated and the level of adipogenic gene expression (PPARγ2 and Adipsin) were higher than those obtained in P2Y₁₃R^+/+ MSC. Interestingly, we observed a significant increase of the number of bone marrow adipocytes in tibia of P2Y₁₃R^-/- mice. In conclusion, our findings indicate that the P2Y₁₃R plays an important role in the balance of osteoblast and adipocyte terminal differentiation of bone marrow progenitors. Therefore, the P2Y₁₃ receptor can be considered as a new pharmacological target for the treatment of bone diseases like osteoporosis. STEM Cells 2013;31:2747–2758