Embryonic stem cells represent a potentially unlimited cell source for tissue engineering applications. However, in order to be used for such applications, embryonic stem cells' differentiation must be controlled to only the desired lineages. In this study, we examine the effects of nanofibrous architecture and biochemical cues on the osteogenic differentiation of embryonic stem cells compared to the more traditional architecture without the nanofibrous features in two dimensions (thin matrix or flat films) and three dimensions (scaffolds) in vitro. After three weeks of culture the nanofibrous thin matrices were capable of supporting mRNA expression of osteogenic differentiation markers in embryonic stem cells without osteogenic supplements, while solid films required osteogenic supplements and growth factors to achieve mRNA expression of osteogenic differentiation markers. Nanofibrous scaffolds substantially enhanced mRNA expression of osteogenic differentiation markers compared to solid-walled scaffolds, nanofibrous thin matrices or solid films. After 4 weeks of culture, nanofibrous scaffolds were found to contain 3 times more calcium and stronger osteocalcin stain throughout the scaffolds than the solid-walled scaffolds. Overall, the nanofibrous architecture enhanced the osteogenic differentiation and mineralization of embryonic stem cells compared to the solid-walled architecture in both two and three-dimensional cultures.