To characterize mesenchymal stem cells (MSC), we compared gene expression profiles in human bone marrow MSC (11 lines) and human fibroblasts (4 lines) by RT-PCR and real time PCR. Messenger RNA levels of MHC-DR-α, MHC-DR-β, MHC-DR-associated protein CD74, tissue factor pathway inhibitor-2, and neuroserpin were much higher in MSC than in fibroblasts, even in the presence of large interindividual variations. Those of adrenomedullin, apolipoprotein D, C-type lectin superfamily member-2, collagen type XV α1, CUG triplet repeat RNA-binding protein, matrix metalloproteinase-1, protein tyrosine kinase-7, and Sam68-like phosphotyrosine protein/T-STAR were lower in MSC than in fibroblasts. FACS analysis showed that cell surface expression of MHC-DR was also higher in MSC than in fibroblasts. MHC-DR expression decreased after osteogenic differentiation, whereas the expression of adrenomedullin—a potent stimulator of osteoblast activity—along with collagen XV α1 and apolipoprotein D increased after osteogenic differentiation. The marker genes identified in this study should be useful for characterization of MSC both in basic and clinical studies.