We show that endothelial cell (EC)-generated vascular guidance tunnels (ie, matrix spaces created during tube formation) serve as conduits for the recruitment and motility of pericytes along EC ablumenal surfaces to facilitate vessel maturation events, including vascular basement membrane matrix assembly and restriction of EC tube diameter. During quail development, pericyte recruitment along microvascular tubes directly correlates with vascular basement membrane matrix deposition. Pericyte recruitment to EC tubes leads to specific induction of fibronectin and nidogen-1 (ie, matrixbridging proteins that link together basement membrane components) as well as perlecan and laminin isoforms. Coincident with these events, up-regulation of integrins, α 5β1, α3β1, α6β1, and α1β 1, which bind fibronectin, nidogens, laminin isoforms, and collagen type IV, occurs in EC-pericyte cocultures, but not EC-only cultures. Integrin-blocking antibodies to these receptors, disruption of fibronectin matrix assembly, and small interfering RNAsuppression of pericyte tissue inhibitor of metalloproteinase (TIMP)-3 (a known regulator of vascular tube stabilization) all lead to decreased EC basement membrane, resulting in increased vessel lumen diameter, a key indicator of dysfunctional EC-pericyte interactions. Thus, pericyte recruitment to EC-lined tubes during vasculogenesis is a stimulatory event controlling vascular basement membrane matrix assembly, a fundamental maturation step regulating the transition from vascular morphogenesis to stabilization.