Although ex vivo expanded mesenchymal stem cells (MSC) have been used in numerous studies, the molecular signature and in vivo distribution status of MSC remain unknown. To address this matter, we identified numerous human MSC‐characteristic genes—including nine transcription factor genes —using DNA microarray and real‐time RT‐PCR analyses: Most of the MSC‐characteristic genes were down‐regulated 24 h after incubation with osteogenesis‐, chondrogenesis‐ or adipogenesis‐induction medium, or 48–72 h after knockdown of the nine transcription factors. Furthermore, knockdowns of ETV1, ETV5, FOXP1, GATA6, HMGA2, SIM2 or SOX11 suppressed the self‐renewal capacity of MSC, whereas those of FOXP1, SOX11, ETV1, SIM2 or PRDM16 reduced the osteogenic‐ and/or adipogenic potential. In addition, immunohistochemistry using antibodies for the MSC characteristic molecules—including GATA6, TRPC4, FLG and TGM2—revealed that MSC‐like cells were present near the endosteum and in the interior of bone marrow of adult mice. These findings indicate that MSC synthesize a set of MSC markers in vitro and in vivo, and that MSC‐characteristic transcription factors are involved in MSC stemness regulation.