Abstract Body (Do not enter title and authors here): Vascular smooth muscle cells and pericytes are mural cells that maintain the integrity and support the function of blood vessels. The molecular mechanism underlying the specification of pericytes and vascular smooth muscle cells remains elusive. Here, we performed spatial transcriptomics analysis of human tissue microvasculature and identified a Notch-Neurotrophin axis in the specification of human mural cells. Mechanistically, NOTCH3 receptor signaling induces Nerve Growth Factor (NGF)-TRKA signaling by transcriptional upregulation of Nerve Growth Factor Receptor (NGFR) expression, which potentiates TRKA signaling, leading to upregulation of pericyte-specific gene expression. Activation of TRKA induces brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT3), leading to upregulation of vascular smooth muscle-specific gene expression and increased cell contractility. In explanted human synovial tissue biopsies containing viable vascular endothelial cells and mural cells, stimulation with NGF, BDNF, or NT3 led to the upregulation of alpha-smooth muscle actin in mural cells and increased vascular wall thickness. In adult mice, systemic administration of neurotrophin receptor agonists induced expression of mural cell marker ACTA2 and vascular smooth muscle cell marker MYH11 in the distal pulmonary arterioles. Our findings suggest neurotrophin signaling downstream of NOTCH3 receptor activation specifies pericyte and vascular smooth muscle cell fate.