Manipulating neural stem cell (NSC) fate is of great importance for improving the therapeutic efficacy of NSCs to treat neurodegenerative disorders. Biophysical cues, in addition to biochemical factors, regulate NSC phenotype and function. In this study, we assessed the extent to which surface nanotopography of culture substrates modulates human NSC (hNSC) differentiation. Fibronectin-coated polymer substrates with diverse nanoscale shapes (groove and pillar) and dimensions (ranging from 300 to 1500 nm groove width and pillar gap) were used to investigate the effects of topographical cues on hNSC morphology, alignment, focal adhesion, and differentiation. The majority of nanopatterned substrates induced substantial changes in cellular morphology and alignment along the patterned shapes, leading to alterations in focal adhesion and F-actin reorganization. Certain types of nanopatterned substrates, in particular the ones with small nanostructures (e.g., 300-300 nm groove ridges and 300-300 nm pillar diameter gaps), were found to effectively enhance focal adhesion complex development. Consequently, these substrates enhanced hNSC differentiation toward neurons and astrocytes. Nanotopographical-induced formation of focal adhesions in hNSCs activates integrin-mediated mechanotransduction and intracellular signaling pathways such as MEK-ERK, which may ultimately promote gene expression related to NSC differentiation. This strategy of manipulating matrix surface topography could be applied to develop culture substrates and tissue engineered scaffolds that improve the efficacy of NSC therapeutics.