Mesenchymal stem cells (MSCs) have the ability to differentiate into a variety of lineages and to renew themselves without malignant changes, and thus hold potential for many clinical applications. However, it has not been well characterized how different the properties of MSCs are depending on the tissue source in which they resided. We previously reported a novel technique for the prospective MSC isolation from bone marrow, and revealed that a combination of cell surface markers (LNGFR and THY-1) allows the isolation of highly enriched MSC populations. In this study, we isolated LNGFR(+) THY-1 (+) MSCs from synovium using flow cytometry. The results show that the synovium tissue contained a significantly larger percentage of LNGFR (+) THY-1 (+) MSCs. We examined the colony formation and differentiation abilities of bone marrow-derived MSCs (BM-MSCs) and synovium-derived MSCs (SYN-MSCs) isolated from the same patients. Both types of MSCs exhibited a marked propensity to differentiate into specific lineages. BM-MSCs were preferentially differentiated into bone, while in the SYN-MSC culture, enhanced adipogenic and chondrogenic differentiation was observed. These data suggest that the tissue from which MSCs are isolated should be tailored according to their intended clinical therapeutic application.