Osteoblasts produce calcified bone matrix and contribute to bone formation and remodeling. In this study, we established a procedure to directly convert human fibroblasts into osteoblasts by transducing some defined factors and culturing in osteogenic medium. Osteoblast-specific transcription factors, Runt-related transcription factor 2 (Runx2), and Osterix, in combination with Octamer-binding transcription factor 3/4 (Oct4) and L-Myc (RXOL) transduction, converted ∼80% of the fibroblasts into osteocalcin-producing cells. The directly converted osteoblasts (dOBs) induced by RXOL displayed a similar gene expression profile as normal human osteoblasts and contributed to bone repair after transplantation into immunodeficient mice at artificial bone defect lesions. The dOBs expressed endogenous Runx2 and Osterix, and did not require continuous expression of the exogenous genes to maintain their phenotype. Another combination, Oct4 plus L-Myc (OL), also induced fibroblasts to produce bone matrix, but the OL-transduced cells did not express Osterix and exhibited a more distant gene expression profile to osteoblasts compared with RXOL-transduced cells. These findings strongly suggest successful direct reprogramming of fibroblasts into functional osteoblasts by RXOL, a technology that may provide bone regeneration therapy against bone disorders.O steoblasts play a central role in bone formation and remodeling by producing type I collagen, osteopontin, osteocalcin, and bone sialoprotein (BSP), and calcifying these bone matrixes (1). They are also involved in hematopoiesis, phosphate metabolism, and glucose metabolism (2). Osteoblasts are derived from mesenchymal progenitor cells that are common precursors shared by chondrocytes, adipocytes, and myoblasts (3). The differentiation of osteoblasts is regulated by various transcription factors, including Runt-related transcription factor 2 [Runx2, also known as core-binding factor subunit α-1 (Cbfα-1)] (4, 5), Osterix (6, 7), Distal-less homeobox 5 (Dlx5) (8), and activation transcription factor 4 (ATF-4) (8). A functional decline in osteoblasts relative to osteoclasts results in imbalance between bone formation and resorption and may cause osteolytic pathological conditions, such as osteoporosis (9), alveolar bone resorption associated with periodontitis (10), and bone lysing associated with bone tumors, including multiple myeloma (11).It has been demonstrated that forced expression of combinations of some transcription factors, such as Octamer-binding transcription factor 3/4 (Oct4), Sox2, Klf-4, and c-Myc (reprogramming factors), induces immortality and pluripotency in mammalian somatic cells (12, 13). The generation of induced pluripotent stem (iPS) cells clearly indicates that genome-wide epigenetic programming can be drastically changed in somatic cells by a small number of transcription factors that may have key regulatory roles in cell fate decisions (14,15).Recent studies have reported that direct conversion, or direct reprogramming, of somatic cells into another dif...