The selective in vitro expansion and differentiation of multipotent stem cells are critical steps in cell-based regenerative therapies, but technical challenges have limited cell yield and thus the success of these potential treatments. The Rho GTPases and downstream Rho kinases (Rho coiled-coil kinases or ROCKs) are central regulators of cytoskeletal dynamics during the cell cycle and thus help determine the balance between stem cells self-renewal, lineage commitment, and apoptosis. Here, we examined if suppression of ROCK signaling enhances the efficacy of bone marrow-derived mesenchymal stem cells (BMSCs) differentiation into neurons and neuroglial cells. BMSCs were cultured in epidermal growth factor (EGF, 10??g/l) and basic fibroblastic growth factor (bFGF, 10??g/l) in the presence or absence of the Rho kinase inhibitor Y-27632 (10??M). The expression levels of neuron-specific enolase (NSE) and glial fibrillary acidic protein (GFAP) were detected by immunofluorescence and Western blotting. The average number of NSE-positive cells increased from 83.20?±?8.677 (positive ratio 0.2140?±?0.0119) to 109.20?±?8.430 (positive ratio 0.3193?±?0.0161) per visual field in the presence of Y-27632, while GFAP-positive cell number increased from 96.30?±?8.486 (positive ratio 0.18?±?0.0152) to 107.50?±?8.683 (positive ratio 0.27?±?0.0115) (P?