Tanaka et al. reported that MYOD1 overAN-9 expression in immature hiPSCs stimulates them to become mature myocytes with very high efficiency and reproducibility. Their method provides relatively uniform undifferentiated cells, which may preclude variation in their differentiation frequency. Their results suggested that obtaining relatively uniform types of cells as early as possible may be very important. We developed a new strategy to purify osteoprogenitors from EB-derived cells by isolating tissue-nonspecific alkaline phosphatase -positive cells using FACS. We found that cells separated from EBs did not express TNAP immediately after single-cell separation. They did not express E-cadherin but expressed relatively high levels of CD90, indicating that they were not progenitors of liver or bile duct epithelial cells. Treating the cells with a combination of transforming growth factor – b, insulin-like growth factor -1, and fibroblast growth factor -2 greatly enhanced TNAP expression. Furthermore, the cells began to express high levels of osterix, which is an exclusive BMS-193885 osteogenic marker. The cells initially expressed low levels of runt-related transcription factor 2, and continuous culture induced high levels of RUNX2, bone sialoprotein, type I collagen, and eventually, osteocalcin. To the best of our knowledge, these are the first observations of osteoprogenitors expressing high levels of TNAP and OSX but low levels of RUNX2 and collagen1a. In general, MSCs in vivo first express RUNX2, which promotes the expression of several early osteogenic marker proteins. These RUNX2-expressing precursors then express OSX and induce differentiation of these cells into mature and functional osteoblasts. Therefore, OSX is a target molecule of RUNX2. However, in our experiment, OSX may have functioned as an initial transcription factor to initiate osteogenesis. We also found that these cells could form multiple mineralized nodules with multidendritic cells that express high levels of receptor activator of NF-kappaB ligand, suggesting they can terminally differentiate into osteocyte-like cells. These cells are easily obtained from iPSCs and are capable of differentiating into osteocyte-like cells; they responded to treatment with activated vitamin D3 by upregulating OCN, providing a new clue in the investigation of osteocytes. iPSCs are powerful tools in many fields of basic scientific research. Several reports have shown that osteogenic cells can be generated from iPSCs.