AbstractMicrovascular homeostasis is strictly regulated, requiring close interaction between endothelial cells and pericytes. Here, we aimed to improve our understanding of how microvascular crosstalk affects pericytes. Human-derived pericytes, cultured in absence, or presence of human endothelial cells, were studied by RNA sequencing. Compared with mono-cultured pericytes, a total of 6704 genes were differentially expressed in co-cultured pericytes. Direct endothelial contact induced transcriptome profiles associated with pericyte maturation, suppression of extracellular matrix production, proliferation, and morphological adaptation. In vitro studies confirmed enhanced pericyte proliferation mediated by endothelial-derived PDGFB and pericyte-derived HB-EGF and FGF2. Endothelial-induced PLXNA2 and ACTR3 upregulation also triggered pericyte morphological adaptation. Pathway analysis predicted a key role for TGFβ signaling in endothelial-induced pericyte differentiation, whereas the effect of signaling via gap- and adherens junctions was limited. We demonstrate that endothelial cells have a major impact on the transcriptional profile of pericytes, regulating endothelial-induced maturation, proliferation, and suppression of ECM production.