Vascular cell functionality is critical to blood vessel homeostasis. Constitutive NF-B activation in vascular cells results in chronic vascular inflammation, leading to various cardiovascular diseases. However, how NF-B regulates human blood vessel homeostasis remains largely elusive. Here, using CRISPR/Cas9-mediated gene editing, we generated RelA knockout human embryonic stem cells (hESCs) and differentiated them into various vascular cell derivatives to study how NF-B modulates human vascular cells under basal and inflammatory conditions. Multi-dimensional phenotypic assessments and transcriptomic analyses revealed that RelA deficiency affected vascular cells via modulating inflammation, survival, vasculogenesis, cell differentiation and extracellular matrix organization in a cell type-specific manner under basal condition, and that RelA protected vascular cells against apoptosis and modulated vascular inflammatory response upon tumor necrosis factor (TNF) stimulation. Lastly, further evaluation of gene expression patterns in IB knockout vascular cells demonstrated that IB acted largely independent of RelA signaling. Taken together, our data reveal a protective role of NF-B/RelA in modulating human blood vessel homeostasis and map the human vascular transcriptomic landscapes for the discovery of novel therapeutic targets.