The study investigates the role of Eph-ephrin signaling in endothelial cell (EC) sorting and arterial specification during retinal development. Using a combination of mouse genetics, in vitro flow experiments, single-cell RNA sequencing, and biochemistry, the researchers found that the balance between ephrin-B2 and its receptor EphB4 is critical for arterial specification, cell sorting, and arteriovenous patterning. Ephrin-B2, which is highly expressed in arteries, and EphB4, which is dominant in veins, interact to control EC fate. Ephrin-B2 promotes arterial specification and migration of tip cell progeny into arteries, while EphB4 represses these processes. The study also revealed that these interactions are influenced by endothelial shear stress and are linked to the transcription factor Dach1. Additionally, the findings suggest that Eph-ephrin signaling integrates cell segregation and arteriovenous specification in the vasculature, which has potential relevance for human vascular malformations caused by *EPHB4* mutations.The study investigates the role of Eph-ephrin signaling in endothelial cell (EC) sorting and arterial specification during retinal development. Using a combination of mouse genetics, in vitro flow experiments, single-cell RNA sequencing, and biochemistry, the researchers found that the balance between ephrin-B2 and its receptor EphB4 is critical for arterial specification, cell sorting, and arteriovenous patterning. Ephrin-B2, which is highly expressed in arteries, and EphB4, which is dominant in veins, interact to control EC fate. Ephrin-B2 promotes arterial specification and migration of tip cell progeny into arteries, while EphB4 represses these processes. The study also revealed that these interactions are influenced by endothelial shear stress and are linked to the transcription factor Dach1. Additionally, the findings suggest that Eph-ephrin signaling integrates cell segregation and arteriovenous specification in the vasculature, which has potential relevance for human vascular malformations caused by *EPHB4* mutations.