Human mesenchymal stem cells (hMSCs) are one of the promising sources for regenerative medicine. In general, hMSCs are supposed to grow indefinitely and differentiate to various kinds of cells. The indefinite growth is concerned due to its misleading to carcinogenesis in the practical use of hMSCs in regenerative medicine. In the present study, we investigated unexpected changes, especially in genetic aspect, in hMSCs during culture utilizing fluorescence in situ hybridization (FISH) with chromosome specific DNA probes for chromosomes 1 and 4, or a locus specific identifier for c-myc. Three lots of hMSCs and three human cancer cell lines (HeLa S3, HOS and OUMS-27) were analyzed. The results of this study suggest that both observation of cell morphology and FISH analysis of c-myc aberrant cells in interphase cells are useful for evaluating safety of hMSCs expanded in vitro.