Mesenchymal stem cells (MSCs) have the multipotent capacity and this potential can be applied for obtaining valuable cell types which can use for cell therapy on various regenerative diseases. However, insufficient availability of cellular source is the major problem in cell therapy field using adult stem cell sources. Recently, human embryonic stem cells (hESCs) have been highlighted to overcome a limitation of adult cellular sources because they retain unlimited proliferation capacity and pluripotency. To use of hESCs in cell therapy, above all, animal pathogen free culture system and purification of a specific target cell population to avoid teratoma formation are required. In this study, we describe the differentiation of a mesenchymal stem cell-like cells population from feeder-free cultured hESCs(hESC-MSCs) using direct induction system. hESC-MSCs revealed characteristics similar to MSCs derived from bone marrow, and undifferentiated cell markers were extremely low in hESC-MSCs in RT-PCR, immunostaining and FACS analyses. Thus, this study proffer a basis of effective generation of specialized human mesenchymal stem cell types which can use for further clinical applications, from xenofree cultured hESCs using direct induction system.