Synthetic extracellular matrices provide a framework in which cells can be exposed to defined physical and biological cues, however no method exists today to manipulate single cells within these matrices to understand fundamental principles of cell migration and define conditions that support or inhibit cell movement. Here, we present a strategy for manipulating individual mammalian stem cells in defined synthetic hydrogels through selective optical activation of Rac. A visible laser was applied to guide directional migration of mammalian stem cells in poly(ethylene glycol), PEG, based hydrogel networks incorporated with an adhesion peptide ligand, Tyr-Arg-Gly-Asp-Ser (YRGDS). Mesenchymal stem cells (MSCs) were transfected with a genetically encoded photoactivatable Rac1 (PA-Rac). Rac is a subfamily of Rho GTPases that plays a pivotal role in cell migration by stimulating actin polymerization.