We analyzed chromatin dynamics and transcriptional activity of human embryonic stem cell (hESC)-derived cardiac progenitor cells (CPCs) and KDR⁺/CD34⁺ endothelial cells generated from different mesodermal origins. Using an unbiased algorithm to hierarchically rank genes modulated at the level of chromatin and transcription, we identified candidate regulators of mesodermal lineage determination. HOPX, a non-DNA-binding homeodomain protein, was identified as a candidate regulator of blood-forming endothelial cells. Using HOPX reporter and knockout hESCs, we show that HOPX regulates blood formation. Loss of HOPX does not impact endothelial fate specification but markedly reduces primitive hematopoiesis, acting at least in part through failure to suppress Wnt/β-catenin signaling. Thus, chromatin state analysis permits identification of regulators of mesodermal specification, including a conserved role for HOPX in governing primitive hematopoiesis.