An in vitro model has been developed to study migration, survival, proliferation, and capillary-like differentiation of human microvascular endothelial cells (HMVECs) in an environment that avoids tumor promoters and complex matrices. HMVEC monolayers were plated, then induced to form three-dimensional, capillary-like networks by overlaying with human type I collagen followed by a second overlay of collagen with embedded fibroblasts. Detachment and migration of endothelial cells into the matrix was triggered within hours by the overlaying collagen, and the fibroblasts stimulated survival and formation of cords, vacuoles, tubes, and, after 4 to 5 days, capillary networks. The differentiation into branching capillary-like structures was dependent on direct fibroblast-endothelial cell contact and was not achieved when fibroblasts were replaced by seven types of melanoma cells, which included radial and vertical growth phase primary and metastatic stages. Vascular endothelial growth factor (VEGF), when overexpressed in fibroblasts, stimulated endothelial cell proliferation and migration, whereas angiopoietin-1 (Ang-1) had only motogenic effects. Neutralizing antibodies against VEGF and blocking antibodies for VEGF-receptor 2 (VEGFR2) significantly inhibited but not completely obliterated capillary network formation, suggesting that the VEGF signaling pathway is important but not exclusive and that other fibroblast-derived soluble factors and fibroblast-endothelial cell contact are essential for endothelial cell survival and differentiation.