Mouse embryonic stem cells (ESC) collect information from their environment and make cell fate decisions based on intrinsic and extrinsic factors. The decision of ESC to differentiate to multiple lineages in vitro occurs during the formation of embryoid bodies (EB). EB formation and ESC fate decisions within EB are influenced by cell-environment interactions. However, the molecular mechanisms underlying cell-environmental modulation of ESC fate decisions are incompletely understood. Since adhesion molecules (AM) influence proliferation and differentiation in developing and adult tissues, we hypothesized that specific AM interactions may guide ESC commitment toward hematopoietic and endothelial lineages. To test this hypothesis, we established that AM in the adherens, tight and gap junction pathways are differentially expressed during early stages of hematopoietic commitment. E-cadherin (E-cad), Claudin-4 (Cldn4), Connexin-43 (Cx43), Zona Occludens-1 (ZO-1) and Zona Occludens-2 (ZO-2) transcript levels were quantified during the commitment process leading to identification of novel markers delineating early hematopoietic and endothelial stages. The functional and phenotypic consequences of modulating AM expression were assessed using hematopoietic colony forming assays, endothelial sprouting assays, and surface protein expression in stable ESC lines with constitutively reduced levels of E-cad, Cldn4, Cx43, ZO-1 and ZO-2. Reduced expression of E-cad, Cx43, Cldn4 and ZO-1 decreased hematopoietic commitment and increased endothelial differentiation approximately 2 fold. Reduction of ZO-1 decreased hematopoiesis approximately 2 fold with minimal influence on endothelial differentiation. These data indicate that a subset of AM influence ESC decisions to commit to endothelial and hematopoietic lineages and suggest that modulation of AM interactions be considered in strategies to guide ESC differentiation for transplantation therapy.