Bone-related studies have been widely carried out by culturing cells on two-dimensional (2D) culture system because of its easiness of handling, but these 2D in vitro achievements may imply a distinct outcome compared with the in vivo situation. On the contrary, three-dimensional (3D) culture system has been suggested as a better biomimetic in vitro model by providing an appropriate cell-cell or cell-matrix interaction. In this study, we successfully reconstructed a 3D disk type of scaffold-free tissue (SFT) using mouse osteoblast-like cells, which evoked an osteocyte differentiation within 2 days. Particularly, the SFT was also utilized as an in vitro osteocytic model to elucidate the effect of hypoxia on cellular differentiation capability. As a result, the hypoxia upregulated a matured osteocyte marker, Sost, in the SFT, whereas both osteoblast and osteocyte markers were significantly downregulated by hypoxia in the 2D conventional monolayer model. The results imply that the hypoxia may enhance the initiation of osteocyte differentiation and retain the osteocyte differentiation in the 3D culture system. Of note, we reported the significance of 3D culture system that might represent the in vivo situation regarding cellular response to stimuli. Hence, our study suggests wide applications of SFT using osteoblast cells as a novel in vitro osteocyte model for the osteocyte-related studies.