The in vitro differentiation potential of rat dental pulp-derived cells into an osteoblastic lineage was examined. Induction was carried out under in vitro osteogenesis-inducing conditions used for mesenchymal stem cells from human bone marrow. We previously reported that the level of mineralization was high at 6 weeks after induction, as determined by von Kossa staining. In the present study, we quantitatively measured the alkaline phosphatase (ALP) activity after induction. The ratios of ALP-positive cells to total cells were 44.4 ± 1.0, 46.9 ± 0.9, 40.5 ± 0.5 and 21.0 ± 1.8% at 1, 2, 3 and 6 weeks after induction, respectively. The level of ALP activity was significantly decreased at 6 weeks after induction (p < 0.05). The stem cell markers STRO-1, SSEA-1, Nanog and Oct-3/4 were expressed in a subset of the cells, suggesting that stem cells were present. To elucidate gene regulation in the osteoblastic lineage of the cells, quantitative gene expression analyses were carried out using real-time RT-PCR. Col1a2 (collagen type I) and Bglap (osteocalcin) were up-regulated by about 1.7-fold and 1.3-fold, respectively, at 6 weeks after induction compared with their corresponding levels at 1 week. These results indicate that the cells differentiated into an osteoblastic lineage in parallel with decreasing ALP activity during induction. Moreover, the expressions of Col1a2 and Bglap were inversely correlated with Alpl (ALP) expression after induction, which was correlated with Bmp2 (bone morphogenetic protein 2) expression. Taken together, the findings indicate that these molecules contribute to the differentiation of rat dental pulp-derived cells into an osteoblastic lineage.