The purpose of this study is to develop strategies to facilitate nerve regeneration using a synergistic combination of guidance cues. We are investigating the cellular mechanisms of development using adult rat hippocampal progenitor cells (AHPCs) and have demonstrated that manipulating a combination of physical, chemical and biological cues can lead to oriented astrocyte and neural progenitor cell outgrowth and can influence progenitor cell differentiation. To provide physical guidance, micropatterned polystyrene (PS) substrates were fabricated and chemically modified with laminin. Astrocytes or AHPCs cultured on these substrates align along the grooves of the patterned surface extending highly elongated processes. To further explore AHPC outgrowth and differentiation, physical guidance cues were integrated with the biological influence of astrocytes. AHPCs co-cultured in contact with astrocytes preferentially acquired neuronal morphology, with nearly double the percentage of cells expressing class III beta-tubulin (TuJ1) on the micropatterned half of the substrate, as opposed to the planar half of the substrate, or compared to those growing in the absence of astrocytes. This indicates that substrate three-dimensional topography, in synergy with chemical (laminin) and biological (astrocytes) guidance cues, facilitates neuronal differentiation of AHPCs. This environment provided biological and spatial control over differentiation enhancing neuronal differentiation and promoting neurite alignment on topographically different regions of the same substrate. In a non-contact co-culture system, astrocyte-derived soluble factors enhanced neurite outgrowth and induced neuronal differentiation with significantly more AHPCs TuJ1 immunoreactive than in the contact co-culture. Therefore, soluble cues may have had a stronger influence on neuronal differentiation and neuritic extension compared to contact mediated factors or a combination of soluble and contact mediated factors that were presented by the monolayer of aligned astrocytes. The results also point to the potential role of localized concentration of these factors within the microgrooves as a reason for the differences in differentiation on micropatterned and planar substrates in the contact as opposed to the non-contact co-cultures. This research provides insights into mechanisms of neural stem cell differentiation and a foundation for the development of a promising nerve regeneration strategy incorporating a synergistic combination of cues for guided central nervous system repair following injury.