The development of methods to induce differentiation of human mesenchymal stem cells (hMSCs) has opened the possibility of using these cells in regenerative or reparative therapies. However, the low frequency of hMSCs in tissue means it is often necessary to expand these cells extensively in vitro. In this study, we evaluated the effects of long-term serial passage on the characteristics of bone marrow-derived hMSC populations. In addition, we examined the effect on subsequent hMSC neural differentiation ability, which has not been reported earlier. The hMSC population examined was found to maintain a stable phenotype during the first 6-8 passages of culture as assessed by proliferative ability, morphological appearance, and surface antigen, gene and protein expression, and also expressed pluripotency and neural lineage markers constitutively in the undifferentiated state. Long-term subcultivation neither resulted in spontaneous neural differentiation nor compromised the ability of hMSCs to develop toward an early neuronal fate. In addition, the transformation elicited in hMSC cultures in response to cytokine-based neuronal differentiation was examined by live cell microscopy. We demonstrated, for the first time, that the observed changes result from active and dynamic processes involving outgrowth and motility of cellular extensions, processes entirely distinct from the rapid epiphenomena of cytotoxicity and cytoskeleton disruption generated by chemical induction methods. Cytokine-induced differentiation of hMSCs was also associated with upregulation of early neural gene and protein expression. These findings support the neuronal differentiation capability of hMSCs, although further investigation is required to confirm the ability to attain a mature neuronal phenotype.