Several studies have demonstrated that three-dimensional (3-D) culture systems influence human embryonic stem cell (hESC) phenotypes and fate choices. However, the effect that these microenvironmental changes have on signaling pathways governing hESC behaviors is not well understood. Here, a 3-D microwell array was used to investigate differences in activation of developmental pathways between 2-D and 3-D cultures of both undifferentiated hESCs and hESCs undergoing initial differentiation in embryoid bodies (EBs). An increased induction into mesoderm and endoderm and differences in expression of genes from multiple signaling pathways that regulate development, including Wnt/beta-catenin, TGF-beta superfamily, Notch, and FGF during EB-mediated differentiation were observed in 3-D microwells as compared with the 2-D substrates. In undifferentiated hESCs, differences in epithelial-mesenchymal transition phenotypes and the TGF beta/BMP pathway between cultures in 3-D and 2-D were also observed. These results illustrate that 3-D culture influences multiple pathways that may regulate the differentiation trajectories of hESCs. (c) 2014 American Institute of Chemical Engineers AIChE J, 60: 1225-1235, 2014