Stem cell microenvironments are enriched by signals from a variety of components, which cooperate spatially and temporally to regulate cellular function. In vitro recapitulating such complexity in a well-controlled manner is elusive. Here, a platform for patterning multiple bio-active proteins on a single substrate is developed and optimized, and is used it to study the cooperative involvement of cell–matrix interaction and cell–cell signaling in regulating neural stem cell (NSC) function. An affinity-capturing-based multi-step microcontact printing is used to pattern, extracellular matrix proteins, and cell–cell signaling ligands, as intersecting lines on a nonadhesive background. Such design provides spatial segregation of signals from different extrinsic components, while allowing cell traffic between them during their proliferation and differentiation processes. Rat embryonic neural stem cells are cultured and characterized on the multicomponent substrates patterned with different combinations of fibronectin, N-cadherin, and Jagged1 proteins and allow to proliferate and differentiate over long term. It is found that local presentation of Notch signaling ligand (Jagged1) or cell adhesion molecule (N-cadherin) effectively modulate the balance between cell–cell and cell–matrix interaction, and significantly change the overall spatial remodeling of NSC differentiation. This platform provides an unambiguous approach to study the spatial and temporal cooperative involvement of different extrinsic components in regulating stem cell behavior. It is also readily expandable for inclusion of extra components and applicable to use with other types of cells, which provide a powerful tool for basic study of cell–material interaction or advanced tissue-interface engineering.