Sprouting angiogenesis is a multi-step process consisting of basement membrane degradation, endothelial cell (EC) activation, proliferation, invasion, lumen formation, and sprout stabilization. Such complexity is consistent with a requirement for orchestration of individual gene expression changes occurs alongside activation of multiple signaling pathways. To better understand the mechanisms that direct the transformation of adherent ECs on the surface of collagen matrices to develop multicellular invading sprouts, we analyzed differential gene expression with time using a defined in vitro model of EC invasion driven by the combination of sphingosine-1-phosphate (S1P), bFGF and VEGF. Gene expression changes were confirmed by real-time PCR and Western blot analyses. A cohort of cell adhesion molecule genes involved in adherens junction and cell-extracellular matrix (ECM) interactions were upregulated, whereas a set of genes associated with tight junctions were downregulated. Numerous genes encoding ECM proteins and proteases were induced, indicating that biosynthesis and remodeling of ECM is indispensable for sprouting angiogenesis. Knockdown of a highly upregulated gene, A Disintegrin and Metalloproteinase with Thrombospondin-type repeats-1 (ADAMTS1), decreased invasion responses, confirming a role for ADAMTS1 in mediating EC invasion. Furthermore, differential expression of multiple members of the Wnt and Notch pathways were observed. Functional experiments indicated that inhibition and activation of the Notch signaling pathway stimulated and inhibited EC invasion responses, respectively. This study has enhanced the molecular roadmap of gene expression changes that occur during endothelial invasion and highlighted the utility of three-dimensional models to study EC morphogenesis.