Microscale topographical features have been known to affect cell behavior. An important target in this area is to integrate top down techniques with bottom up self-assembly to create three-dimensional (3D) patterned bioactive mimics of extracellular matrices. We report a novel approach toward this goal and demonstrate its use to study the behavior of human mesenchymal stem cells (hMSCs). By incorporating polymerizable acetylene groups in the hydrophobic segment of peptide amphiphiles (PAs), we were able to micro-pattern nanofiber gels of these bioactive materials. PAs containing the cell adhesive epitope arginine-glycine-aspartic acid-serine (RGDS) were allowed to self-assemble within microfabricated molds to create networks of either randomly oriented or aligned ∼30 nm diameter nanofiber bundles that were shaped into topographical patterns containing holes, posts, or channels up to 8 m in height and down to 5 m in lateral dimensions. When topographical patterns contained nanofibers aligned through flow prior to gelation, the majority of hMSCs aligned in the direction of the nanofibers even in the presence of hole microtextures and more than a third of them maintained this alignment when encountering perpendicular channel microtextures. Interestingly, in topographical patterns with randomly oriented nanofibers, osteoblastic differentiation was enhanced on hole microtextures compared to all other surfaces.