The endothelial cell protein C receptor (EPCR) is expressed by endothelial cells of large blood vessels and by hematopoietic stem cells. DNaseI hypersensitive (DH) site mapping across 38 kb of the human EPCR gene (hEPCR) locus identified 3 potential regulatory elements. By itself, the DH region spanning the proximal promoter (PP) was unable to direct cell-specific transcription in transgenic mice. A second DH element, located upstream of PP and termed -5.5HS was hypersensitive only in endothelial cells (ECs) and immature hematopoietic cell lines. Transgenes expressing LacZ under the control of -5.5HS coupled to either PP or the SV40 promoter were able to direct beta-galactosidase activity to the endothelium of large vessels during embryogenesis and adulthood. The -5.5HS exhibited enhancer activity that was conferred by the interplay of transcription factors interacting with conserved Ets and composite GATA/Tal1 motifs. The third DH element, located in intron 2, was primarily hypersensitive in EPCR-negative cells, and capable of initiating antisense transcription, suggesting a role in hEPCR silencing. This study identifies critical elements required for the tissue specificity of hEPCR and suggests a mechanism for endothelial and hematopoietic stem cell-specific transcriptional regulation that reflects the common origin of these cell types.