Stem cell technology combined with nano-scaffold surfaces provides a new tool for better induction involved in cell lineage differentiations and therefore for central nervous system repair. This study was undertaken to investigate appropriate neural cell-substrate interactions. Neural progenitors (NPs) were established from human embryonic stem cells (hESCs), as a first step, using an adherent system and a defined medium supplemented with a combination of factors. Next, the behavior of hESC-derived NPs (hESC-NPs) was evaluated on a synthetic, randomly oriented, three-dimensional nanofibrillar matrix composed of electrospun polyamide nanofibers (Ultra-Web™) using a variety of experimental approaches. We have demonstrated that homogenous, expandable, and self-renewable NPs can be easily generated from hESCs; they can express related markers Nestin, Sox1, and Pax6; and they can undergo multipotency differentiation to neurons and glials. Functionally, NPs cultured on nanofibers demonstrated an increase in the rate of migration, proliferation, morphology, and neurite length when compared with NPs cultured on two-dimensional culture surfaces. The results suggest that topographical features of the extracellular matrix of the cell environment have paved the way for a better understanding of human neuronal development, thus allowing for future clinical applications.