Tissue engineering has been studied as a novel therapeutic technology which replaces organ transplantation. Tissue engineering consists of three factors “scaffolds”, “cells” and “growth factors”, and regenerates defecting tissue using them. We have successfully developed porous apatite-fiber scaffolds (AFSs) which have three-dimensional (3D) inter-connected pores using single-crystal apatite fibers and carbon beads; subsequently, we have clarified that the AFSs have an excellent bioactivity on the basis of both in vitro and in vivo evaluations. In addition, we have reconstructed the 3D tissue-engineered bone through 3D-cell culture of mesenchymal stem cells derived from rat bone marrow (RBMC) using the AFS settled into the radial-flow bioreactor (RFB). Aim in the present work is to examine the effect of flow rate of medium in the RFB on the differentiation of osteoblasts in tissue-engineered bone reconstructed using an AFS and RBMC. The flow rates were set to 1.3 and 6.3 cm3∙min-1; tissue-engineered bones reconstructed by the two flow rates are defined as “bone#1” and “bone#2”, respectively. The ALP activity and OC amount normalized for DNA content of bone#2 were higher than those of bone#1. These results indicate that the faster flow rate may promote the differentiation into osteoblast. Thus, the physical irritation to cells, such as flow rates, may be effective for reconstruction of tissue-engineered bone.