A major technological barrier to large-scale propagation of human embryonic stem (HES) cells is the persistence of spontaneous differentiation in culture. Our laboratory and others have previously reported that substrate topography, independent of surface chemistry, profoundly modulates fundamental cell behaviors. We hypothesized that topographic cues would also play a role in modulating HES cell behaviors. This hypothesis was tested on substrates containing nanoscale through micron scale grooves and ridges that were generated by soft lithography. Topographically patterned substrates improved maintenance of the self-renewing phenotype (p = 6.7x10-6) under culture conditions that promote stem cell self-renewal. Topographic cues were found to promote differentiation, however, under culture conditions that promote differentiation. To our knowledge these are the first experiments documenting that the physical topography of culture surfaces influences HES cell differentiation and self-renewal. Topographic cues should be considered a fundamental environmental factor that has relevance to emerging strategies of stem cell engineering.