Eph-ephrin signaling couples endothelial cell sorting and arterial specification. Eph-ephrin signaling is critical for cell sorting and arteriovenous (AV) patterning. The balance between ephrin-B2 and its receptor EphB4 is essential for arterial specification, cell sorting, and AV patterning. Eph-ephrin interactions intersect with VEGF-mediated signaling and act both upstream and downstream of the Notch pathway. Ephrin-B2, which is strongly upregulated after loss of EphB4, promotes major signaling responses regulating cell sorting, migration under flow, and the specification of arterial identity. EphB4 and ephrin-B2 promote arteriovenous segregation. EphB4 restrains the incorporation of tip cell progeny into arteries, whereas ephrin-B2 promotes this process. EphB4 and ephrin-B2 regulate EC heterogeneity and cell fate plasticity. EphB4 and ephrin-B2 modulate HUVEC heterogeneity and cell fate plasticity. EphB4 and ephrin-B2 regulate EC function and AV fate specification. EphB4 and ephrin-B2 control the equilibrium of AV markers and angiogenic regulators in ECs. EphB4 and ephrin-B2 interact with SoxF transcription factors. EphB4 and ephrin-B2 regulate VEGFR2 activation and signaling. EphB4 limits VEGFR2 activation, signaling, and turnover. EphB4 and ephrin-B2 direct the progeny of Esm1+ cells into growing arteries. EphB4 and ephrin-B2 interact with Notch and ERK signaling. EphB4-controlled arterial specification initiates cell cycle exit. Eph-ephrin function is linked to the transcription factor Dach1. Flow sensing is an important mediator of AV specification and is necessary for the maintenance of arterial identity. Loss of blood flow induces defects that can lead to AV malformations. Arterial ECs and their nuclei are elongated and align along the flow axis in the postnatal retinal vasculature. Ephrin-B2 and EphB4 play opposite roles in artery formation. EphB4 and ephrin-B2 regulate EC segregation and artery formation. EphB4 and ephrin-B2 regulate the expression of arterial and venous genes. EphB4 and ephrin-B2 regulate the expression of SoxF transcription factors. EphB4 and ephrin-B2 regulate the expression of VEGFR2 and ERK signaling. EphB4 and ephrin-B2 regulate the expression of Dach1. EphB4 and ephrin-B2 regulate the expression of Notch and ERK signaling. EphB4 and ephrin-B2 regulate the expression of cell cycle regulators. EphB4 and ephrin-B2 regulate the expression of arterial and venous markers. EphB4 and eEph-ephrin signaling couples endothelial cell sorting and arterial specification. Eph-ephrin signaling is critical for cell sorting and arteriovenous (AV) patterning. The balance between ephrin-B2 and its receptor EphB4 is essential for arterial specification, cell sorting, and AV patterning. Eph-ephrin interactions intersect with VEGF-mediated signaling and act both upstream and downstream of the Notch pathway. Ephrin-B2, which is strongly upregulated after loss of EphB4, promotes major signaling responses regulating cell sorting, migration under flow, and the specification of arterial identity. EphB4 and ephrin-B2 promote arteriovenous segregation. EphB4 restrains the incorporation of tip cell progeny into arteries, whereas ephrin-B2 promotes this process. EphB4 and ephrin-B2 regulate EC heterogeneity and cell fate plasticity. EphB4 and ephrin-B2 modulate HUVEC heterogeneity and cell fate plasticity. EphB4 and ephrin-B2 regulate EC function and AV fate specification. EphB4 and ephrin-B2 control the equilibrium of AV markers and angiogenic regulators in ECs. EphB4 and ephrin-B2 interact with SoxF transcription factors. EphB4 and ephrin-B2 regulate VEGFR2 activation and signaling. EphB4 limits VEGFR2 activation, signaling, and turnover. EphB4 and ephrin-B2 direct the progeny of Esm1+ cells into growing arteries. EphB4 and ephrin-B2 interact with Notch and ERK signaling. EphB4-controlled arterial specification initiates cell cycle exit. Eph-ephrin function is linked to the transcription factor Dach1. Flow sensing is an important mediator of AV specification and is necessary for the maintenance of arterial identity. Loss of blood flow induces defects that can lead to AV malformations. Arterial ECs and their nuclei are elongated and align along the flow axis in the postnatal retinal vasculature. Ephrin-B2 and EphB4 play opposite roles in artery formation. EphB4 and ephrin-B2 regulate EC segregation and artery formation. EphB4 and ephrin-B2 regulate the expression of arterial and venous genes. EphB4 and ephrin-B2 regulate the expression of SoxF transcription factors. EphB4 and ephrin-B2 regulate the expression of VEGFR2 and ERK signaling. EphB4 and ephrin-B2 regulate the expression of Dach1. EphB4 and ephrin-B2 regulate the expression of Notch and ERK signaling. EphB4 and ephrin-B2 regulate the expression of cell cycle regulators. EphB4 and ephrin-B2 regulate the expression of arterial and venous markers. EphB4 and e