The Notch family of cell-surface receptors has been proposed to regulate cell-fate decisions by modulating the ability of each cell to respond to environmental cues. In vertebrates, gain-of-function and loss-of-function studies have demonstrated a requirement for Notch signaling for proper patterning of the vasculature during embryogenesis. To examine the molecular mechanisms by which Notch regulates vascular development, we analyzed changes in gene expression in response to Notch signaling. Notch signal transduction and function were assessed in primary human endothelial cells isolated from the dermal microvasculature of neonates, HMVECd. We demonstrate that HMVECd cells express a heterodimeric form of endogenous Notch4 on their cell surface. Using an in vitro coculture assay, we found that Delta4 can function as a ligand for Notch4 in HMVECd cells. Moreover, ectopic expression of an activated allele of Notch4 upregulated the expression of Delta4, suggesting that there may be a regulatory loop between Notch4 and its ligand, Delta4. Notch4 activation also induced the expression of the transcriptional repressors, HES1, HERP1, and HERP2, as well as ephrinB2, an angiogenic factor proposed to be involved in arterial/venous endothelial cell specification.