<p>Osteoblast growth and differentiation, which are controlled by Ca²⁺ signaling, are important for bone formation and homeostasis. It has been reported that the inward rectifier Kir2.1K⁺ channel is responsible for osteoblast differentiation by maintaining the resting membrane potential to regulate intracellular Ca²⁺ concentration ([Ca²⁺]_i). ATP released from hemichannels, such as pannexins and connexins located within the cell membrane, also contributes to osteoblast differentiation. The purpose of this study is to elucidate the functional relationship between Kir2.1 and ATP signaling in osteoblast differentiation of the osteoblastic cell line MC3T3-E1, established from mouse calvaria. We found that the alkaline phosphatase ALP mRNA expression in MC3T3-E1 cells and the endochondral ossification of metatarsal bones were suppressed by the treatment with ML133, a Kir2.1 inhibitor or CBX, a hemichannel blocker. The pharmacological blockade of hemichannels or Kir2.1 also decreased the amount of released ATP from MC3T3-E1 cells during osteoblast differentiation. ATP stimulation induced a transient Ca²⁺ influx through ATP-sensitive Ca²⁺ permeable channels, and this ATP-stimulated [Ca²⁺]_i rises were attenuated by the treatment with ML133 and 5-BDBD, a P2X4 inhibitor. These results suggest that both Kir2.1 and hemichannels may be involved in ATP signaling during osteoblast differentiation. The functional coupling between Kir2.1 and P2X4 channels plays an essential role in maintaining the bone homeostasis via modulating osteoblast differentiation.</p>