SUMMARY Approaches to vascular regeneration and repair require specification of endothelial cells that are competent to form functioning blood vessels. However, the process by which endothelial cells are specified from mesoder- mal precursors remains poorly understood. A key transcriptional regulator of endothelial cell (EC) specification is the ETS-family transcription factor ETV2. Our preliminary data shows that mesodermal progenitor cells (MPCs), differentiated from human induced pluripotent stem cells (iPSCs), are rapidly and efficiently repro- grammed by ETV2 into endothelial cell-like cells (iEC-Ms). These iEC-Ms exhibit properties of endothelial cells in vitro, and assemble into perfused vascular networks in the in vivo microvascular graft assay. In contrast, ETV2 expression directly in iPSCs yielded cells that expressed endothelial cell markers (iEC-Ps) and exhibited a subset of endothelial cell properties in vitro, but did not form perfused vascular networks in microvascular grafts. The overarching goal of this proposal is to use this experimental paradigm to define the mechanisms by which ETV2 drives reprogramming to iECs, and to dissect the mechanisms by which the starting cell type (MPC vs iPSCs) influences the functional properties of the resulting iECs. We propose 3 Specific Aims to achieve these goals: (1) To dissect the transcriptional regulatory landscape of endothelial differentiation. (2) To determine the molecular mechanisms that limit functionality of iECs differ- entiated directly from iPSCs. (3) To characterize the protein-protein interactions required for ETV2 to drive iEC specification. To achieve these aims, we will use cutting edge technologies including single cell RNA-seq, ChIP-seq, and proximity proteomics. Together, these studies will define the molecular mechanisms that underlie the earliest stages of endothe- lial cell specification and that establish endothelial cell competence for interaction with support cells and forma- tion of functional vessels. This fundamental knowledge will form the foundation for strategies to promote vessel development in organ repair and regeneration.