Osteocytes and osteoblasts form a three-dimensional (3D) cellular network in bone. Cellular network has important roles in mechanosensation and mechanotransduction. Previous studies indicated that fluid shear stress was specifically delivered to bone surface and enhanced the activation of the autonomous intracellular Ca2+ ([Ca2+]i) oscillations in osteoblasts and osteocytes via gap junction-mediated cell-cell communication. However the molecular mechanisms in the 3D environment are poorly understood. Here we observed 3D time-lapse autonomous and flow induced Ca2+ signaling in osteocytes and analyzed [Ca2+]i mobilization of osteoid-osteocytes and mature osteocytes in chick calvaria. [Ca2+]i was monitored using a calcium indicator probe, Fluo-8 and analyzed in real time. In response to the flow, [Ca2+]i significantly increased in mature osteocytes in comparison with osteoid-osteocytes. Furthermore, to investigate the differences in response between mature osteocytes and osteoid-osteocytes in detail, we used osteocyte-like cell line, MLO-Y4 which were 3D cultured within type I collagen gels. To study the changes over time in the mRNA expression of osteocyte marker genes, the Sost and Dmp1 during differentiation of MLO-Y4 cells were studied in vitro, mRNA samples were collected on 7 days and 15 days and evaluated for the gene expression. On day 15, Sost mRNA level was increased in comparison with day 7, whereas Dmp1 mRNA expression was remarkably decreased. These results indicated that MLO-Y4 cells differentiated into mature osteocyte-like cells by long term culture. To assess the functional changes with the osteocyte differentiation, the expression of osteocyte related genes were examined. As a result, the Cx43, c-Fos and Col1a1 mRNA expression were significantly increased on day 15 in comparison with day 7. These findings collectively indicate that [Ca2+]i mobilization is increased by the formation of gap junctional intracellular communication with the differentiation of osteocytes. These effects regulate mineralization-related genes in the osteocyte lineage.