Stem cell microenvironments are enriched by signals from a variety of components,which cooperate spatially and temporally to regulate cellular function [1].In vitro recapitulating such complexity in a well-controlled manner has been elusive.Here,we developed and optimized a platform for patterning multiple bio-active proteins on a single substrate,and used it to study the cooperative involvement of cell-matrix interaction and cell-cell signaling in regulating neural stem cell (NSC) functions.An affinity-capturing based multi-step microcontact printing was used to pattern,extracellular matrix proteins and cell-cell signaling ligands,as intersecting lines on a non-adhesive background.Such design provides spatial segregation of signals from different extrinsic components; while allows cells traffic between them during their proliferation and differentiation processes.Rat embryonic neural stem cells were cultured and characterized on the multicomponent substrates patterned with different combinations of fibronectin,N-cadherin and Jagged1 proteins and allowed to proliferate and differentiate over long term.We 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 regulation mechanisms of multiple components in stem cell microenvironment,is readily expandable for inclusion of extra components and applicable to use with other types of cells,which would provide a powerful tool for basic cell-material interaction studies or advanced tissue-interface engineering,and provide valuable guidance for designing novel biomaterials for stem cell based clinical applications.