Considerable progress has occurred toward our understanding of the molecular basis for vascular morphogenesis, maturation, and stabilization. A major reason for this progress has been the development of novel in vitro systems to investigate these processes in 3D extracellular matrices. In this chapter, we present models of human endothelial cell (EC) tube formation and EC-pericyte tube co-assembly using serum-free defined conditions in 3D collagen matrices. We utilize both human venous and arterial ECs and show that both cell types readily form tubes and induce pericyte recruitment and both ECs and pericytes work together to remodel the extracellular matrix environment by assembling the vascular basement membrane, a key step in capillary tube network maturation and stabilization. Importantly, we have shown that these events occur under serum-free defined conditions using the hematopoietic stem cell cytokines, SCF, IL-3, and SDF-1α and also including FGF-2. In contrast, the combination of VEGF and FGF-2 fails to support vascular tube morphogenesis or pericyte-induced tube maturation under the same serum-free defined conditions. Furthermore, we present novel assays whereby we have developed both human ECs and pericytes to induce specific genes using a doxycycline-regulated lentiviral system. In this manner, we can upregulate the expression of wild-type or mutant gene products at any stage of vascular morphogenesis or maturation in 3D matrices. These in vitro experimental approaches will continue to identify key molecular requirements and signaling pathways that control fundamental events in tissue vascularization under normal or pathologic conditions. Furthermore, these models will provide new insights into the development of novel disease therapeutic approaches where vascularization is an important pathogenic component and create new ways to assemble capillary tube networks with associated pericytes for tissue engineering applications.