P2 receptors activated by ATP are expressed in the skeletal system. However, the role of P2 receptors in osteoblast differentiation remains unclear. Methods: Participation of P2 receptors in differentiation was investigated in the preosteoblast MC3T3-M1 cell line. Preosteoblasts were stimulated for 7 or 14 days in the presence of osteogenic medium containing ATP and its analogs, and then alkaline phosphatase (ALP) activity, gene expression analyses, and protein expression were assessed. Results: We observed that ATP and its analogs promoted increased ALP activity after 7 days of treatment. In contrast, these agonists promoted reductions in ALP activity after 14 days. Some antagonists, such as PPADS (P2 antagonist), MRS2179 (P2Y1 antagonist), MRS2578 (P2Y6 antagonist), and AZ11645373 (P2X7 antagonist) reduced the increases in ALP activity after 7 days. However, only AZ11645373 inhibited the reduction in ALP activity after 14 days. The expression of the P2Y2, P2Y6, P2X4, and P2X7 receptors was observed. Furthermore, treatment with ATP modulated the expression of P2 receptors, increasing P2X4 expression and reducing P2Y6 and P2X7 expression. Similar results were observed after 14 days. In addition, ATP treatment for 7 days increased the expression of transcription factors associated with osteoblast differentiation, such as Runx2, SP7, and Dix5, whereas SP7 and Dix5 expression was reduced at 14 days. These results suggest that P2 receptor activation modulates the differentiation of osteoblasts and is dependent upon the stage of differentiation. These results also suggest that several P2 receptors are involved in this process.