The ability to generate neural lineages from human embryonic stem cells (hESCs) in a controlled manner would further investigation of human neurogenesis and development of potential cell therapeutic applications to treat neurological diseases; however, generating such neural stem cells (NSCs) remains a challenge. In an attempt to characterize the cellular mechanisms involved in hESC differentiation into neuroprogenitor cells, we performed 2-DE using protein extracts from hESC-derived embryoid bodies (EBs) and neuroectodermal spheres (NESs) bearing neuroprogenitors. Of 47 differentially expressed protein spots, 28 nonredundant spots were shown to be upregulated in the NESs; these protein spots included neurogenesis-related proteins (TAF1, SEPT2, NPH3, and CRABP), as expected. Interestingly, 6 of these 28 protein spots were cytoskeleton-associated proteins (CSAP) such as Fascin-1, Cofilin-1, and Stathmin-1. Western-blot analyses confirmed the increased levels of these proteins in the NESs. Furthermore, immunostaining analysis showed that both Fascin-1 and Stathmin-1 were preferentially expressed in the inner rims of neural rosettes, which are characteristic features of neuroprogenitors in culture. We also confirmed prominent expression of Fascin-1 in (sub-)ventricular zone in E15.5 mouse fetal brain. Our results suggest that, in addition to the induction of those genes involved in neural development, hESC differentiation into the NES is associated with a marked reorganization of the cellular cytoskeleton.