Stem cells are defined by their capacities for self-renewal and differentiation into one or more cell lineages, and these processes are regulated by signals from the stem cell microenvironment, or niche, in various tissues throughout organismal development and adulthood. These niches control cell function by presenting complex, regulatory signals including soluble small molecules and proteins, extracellular matrix (ECM) signals, and mechanical cues. There has been considerable progress in studying soluble signal regulation of stem cell function, but comparatively less work has been focused on investigating the “solid phase” of the microenvironment, in large part due to experimental complexities in studying large matrix and other proteins. Recent work demonstrates that bioactive, synthetic materials (i.e., matrices, scaffolds, or culture substrates) can be harnessed to emulate and thereby study the effects of solid phase, or biophysical, signals on stem cell function. We have harnessed biomaterials to investigate a number of aspects of the stem cell microenvironment and have found that extracellular mechanical properties, or stiffness, can profoundly impact neural stem cell self-renewal and differentiation into neurons vs. glia, and mechanistic analysis implicates Rho GTPases as key “mechanotransducers” in this process. Furthermore, immobilization of biochemical signals in the solid phase of a natural niche, including extracellular matrix motifs as well as matrix-binding growth factors, can lead to nanoscale organization of these signals that can modulate signal transduction by controlling cellular receptor clustering. We have developed a hybrid system composed of growth factors oligomerized with a synthetic material and find that nanoscale organization of signaling molecules can profoundly modulate their bioactivity and potency, for example in regulating the proliferation of neural stem cells and the neuronal differentiation of human embryonic stem cells. Finally, we have found that the combinatorial presentation of different motifs from individual extracellular matrix proteins from a material can generate synthetic systems capable of supporting the self-renewal and differentiation of both neural stem cells and human embryonic stem cells, thereby enabling the dissection of the ECM into key individual signals necessary to support stem cell function. Biomimetic materials therefore represent systems that can be used to study principles by which the solid phase of a stem cell microenvironment regulates cell function, as well as offer safe and scaleable systems to precisely control stem cell function for biotechnological and biomedical application.