We present a method for patterning neural stem cells based on pre-patterning polypeptides on a cell-repellent surface (poly(ethylene) oxide-like, PEO-like, plasma-deposited films). The method ensures cell attachment and stability for several weeks, as well as it allows cell migration and differentiation. Various patterns of approximately 1 nm thick cell adhesive poly-L-lysine (PLL) have been created on a cell-repellent PEO-like matrix by microcontact printing using different array configurations and printing conditions. The cell-repellent property of PEO-like film determined the confinement of the cells on the printed patterns. Optimization of the printing method showed that the most homogeneous patterns over large areas were obtained using PLL diluted in carbonate buffer (100mM) at pH 8.4. Neural stem cells cultured on the PLL patterns in low serum and in differentiating medium over 20 days exhibited a good confinement to the polypeptide domains. The number of cells attached increased linearly with the micro-stamped PLL area. The cells were able to extend random axon-like projections to the outside of the patterns and presented high amount of ramifications when cultured in differentiating medium. Migration and axon-like outgrowth have been successfully guided by means of an interconnected squares configuration. The surfaces are suitable for controlling the patterning of stem cells and provide a platform for the assessment of the way how different cell arrangements and culture conditions influence cell interactions and cell developmental processes.