Notch is a transmembrane protein that plays a critical role in the determination of cellular differentiation pathways. Although its importance in the development of mesenchymal tissues has been suggested, its role in skeletal tissues has not been well investigated. Northern blot experiments showed the expression of Notch1 in MC3T3-E1 osteoblastic cells at early differentiation stages. When a Notch1 cytoplasmic domain (Notch-IC [NIC]) delivered by an adenovirus vector was expressed in osteoblastic MC3T3-E1 cells, a significant increase in calcified nodule formation was observed in long-term cultures. Activation of endogenous Notch in MC3T3-E1 by coculturing them with Delta-like-1 (Dll1)-expressing myeloma cells also resulted in a stimulation of calcified nodule formation. Not only affecting nodule formation, Notch activation also had effects on osteoblastic differentiation of multipotent mesenchymal cells. Osteoblastic differentiation of C3H10T1/2 cells induced by bone morphogenetic protein 2 (BMP-2) was significantly stimulated, whereas adipogenic differentiation was suppressed strongly, resulting in a dominant differentiation of osteoblastic cells. NIC expression in primary human bone marrow mesenchymal stem cells (hMSCs) also induced both spontaneous and stimulated osteoblastic cell differentiation. These observations suggest that osteoblastic cell differentiation is regulated positively by Notch and that Notch could be a unique and interesting target molecule for the treatment of osteoporosis.