The formation of a microvasculature is regulated in large part by cell-cell interactions. Ephrins and their Eph receptors mediate cell adhesion, repulsion, and migration, all critical processes in angiogenesis. (1) Here we use a covalently immobilized ephrinA1, conjugated to poly(ethylene glycol), to induce vessel formation both in vitro and in vivo in poly(ethylene glycol) diacrylate (PEGDA) hydrogels. Human umbilical vein endothelial cell (HUVEC) tubulogenesis in matrix metalloproteinase-sensitive hydrogels was visualized from 6 h to 7 days in response to three different concentrations of PEG-ephrinA1. The deposition of extracellular matrix proteins collagen IV and laminin that stabilize tubule formation were imaged, quantified, and found to be dependent on PEG-ephrinA1 concentration. To confirm the importance of the EphA2-ephrinA1 interaction in tubule formation, soluble EphA2 was used to disrupt the EphA2-ephrinA1 interaction between a coculture of HUVEC and human brain vascular pericyte cells. HUVECs seeded onto PEGDA hydrogels displayed a dose-dependent reduction in tubule formation in response to the soluble EphA2. Finally, hydrogels with releasable platelet-derived growth factor (PDGF), immobilized RGDS, and covalently immobilized PEG-ephrinA1 were implanted into the mouse cornea micropocket. These hydrogels induced a more robust vascular response with an increase in vessel density as compared with hydrogels with releasable PDGF alone. As such, PEG-ephrinA1 may represent a promising molecule to regulate cell adhesion and migration for formation of a microvasculature in tissue-engineered constructs.