Bone marrow multipotent stromal cells (or mesenchymal stem cells; MSCs) have the capacity for renewal and the potential to differentiate in culture into several cell types including osteoblasts, chondrocytes, adipocytes, cardiomyocytes, and neurons. This study was designed to investigate the protein expression profiles of rat bone marrow MSCs during differentiation into adipogenic (by dexamethasone, isobutylmethylxanthine, insulin, and indomethacin), cardiomyogenic (by 5-azacytidine), chondrogenic (by ascorbic acid, insulin-transferrin-selenous acid, and transforming growth factor-β1), and osteogenic (by dexamethasone, β-glycerophosphate, and ascorbic acid) lineages by well-known differentiation inducers. Proteins extracted from differentiated MSCs were separated using two-dimensional gel electrophoresis (2-DE) and protein spots were detected using Sypro Ruby dye. Protein spots that were determined to be up- or down-regulated when the expression of corresponding spots (between weeks 1 and 2, 1 and 3, 1 and 4) showed an increase (≥2-fold) or decrease (≤0.5-fold) were successfully identified by MALDI-TOF-MS. In summary, 23 new proteins were identified either up- or down-regulated during differentiation experiments.