Mesenchymal stem cells (MSCs) have been isolated from many tissues including differentiating human embryonic stem cells (hESCs). Derivation of MSCs from hESCs consists of two major steps: differentiation and isolation. In our hands, differentiation of hESCs towards MSC-enriched culture can be induced by trypsinizing hESCs into single cells and plating them on gelatin-coated plates in a culture condition that enhances survival of hESC-derived MSCs and not hESCs. The trypsinized hESCs were grown with feeder support and the medium was supplemented with basic fibroblast growth factor (FGF2) and platelet-derived growth factor (PDGF)-AB. A highly enriched MSC culture could be obtained by repeated passaging by trypsinization. The enriched MSC cultures could be further purified by limiting dilution or FACS sorting for CD105(+) or CD73(+) and CD24(-).