An understanding of the pathways responsible for differentiation in pluripotent stem cells (PSCs) would accelerate their translation to medical therapies. Specifically, studies that identify criteria for the better design of experiments targeting certain phenotypes would allow for the generation of cell sources adequate for transplantation. In this dissertation, we aimed at elucidating the role of the cytoskeleton in the spontaneous differentiation of PSCs in two dimensional (2D) and three dimensional (3D) microenvironments. First, we quantified the expression of the cytoskeleton in ESCs, iPSCs, and the iPSC source phenotype, showing that there were indeed differences in the expression of microfilaments and certain intermediate filaments among all three phenotypes. Next, we found that there were inherent differences in ESC differentiation when cultured in 2D and 3D microenvironments. Lastly, alterations in the cytoskeleton were found to decrease mesodermal differentiation in 3D culture, while increase both mesodermal and endodermal differentiation in 2D culture. Taken together, we identified the cytoskeleton as a regulator of differentiation to the mesodermal and endodermal lineages in both 2D and 3D culture.