Considerable progress has been made toward a molecular understanding of how cells form lumen and tube structures in three-dimensional (3D) extracellular matrices (ECM). This progress has occurred through work performed with endothelial and epithelial cell models using both in vitro and in vivo approaches. Despite the apparent similarities between endothelial and epithelial cell lumen and tube formation mechanisms, there are clear distinctions that directly relate to their functional differences. This review will focus on endothelial cell (EC) lumen formation mechanisms which control blood vessel formation during development and postnatal life. Of great interest is that an EC lumen signaling complex has been identified which controls human EC lumen and tube formation in 3D matrices and which coordinates integrin-ECM contacts, cell surface proteolysis, cytoskeletal rearrangements, and cell polarity. This complex consists of the collagen-binding integrin α2β1, the collagen-degrading membrane-type 1 matrix metalloproteinase (MT1-MMP), junction adhesion molecule (Jam)C, JamB, polarity proteins Par3 and Par6b, and the Rho GTPase Cdc42-GTP. These interacting proteins are necessary to stimulate 3D matrix-specific signaling events (including activation of protein kinase cascades that regulate the actin and microtubule cytoskeletons) to control the formation of EC lumens and tube networks. Also, EC lumen formation is directly coupled to the generation of vascular guidance tunnels, enzymatically generated ECM conduits that facilitate EC tube remodeling and maturation. Mural cells such as pericytes are recruited along EC tubes within these tunnel spaces to control ECM remodeling events resulting in vascular basement membrane matrix assembly, a key step in tube maturation and stabilization.