In this study, we developed an extracellular matrix culture system for increasing endothelial differentiation and ensuring animal free hESC-ECs. We designed a staged protocol that involved EB formation (stage 1) and expansion of endothelial lineage by subculturing EBs in collagen (stage 2). We then derive a highly pure endothelial population by CD31/CD144 double sorting using flow cytometry. In order to define at a molecular level the changes occurring at each stage of hESC differentiation to endothelial cell progeny, and to validate that these cells are similar to human umbilical vein endothelial cells (HUVECs), we also perform transcriptional profiling using whole human genome microarrays and real-time PCR arrays. Finally, to fully understand the beneficial effects of stem cell therapy, one must also be able to track the transplanted cells in living subjects over time in order to better understand their behavior and function in vivo. Therefore, we performed multi-modality imaging in a murine dorsal window model and a murine myocardial ischemia model to assess hESC-EC fate and function.