EPHB4 is a receptor protein tyrosine kinase that is required for the development of lymphatic vessel (LV) valves. We show here that EPHB4 is necessary for the specification of LV valves, their continued development after specification, and the maintenance of LV valves in adult mice. EPHB4 promotes LV valve development by inhibiting the activation of the Ras-MAPK pathway in LV endothelial cells (LEC). For LV specification, this role for EPHB4 depends on its ability to interact physically with the p120 Ras-GTPase-activating protein (RASA1) that acts as a negative regulator of Ras. Through physical interaction, EPHB4 and RASA1 dampen oscillatory shear stress (OSS)-induced Ras-MAPK activation in LEC, which is required for LV specification. We identify the Piezo1 OSS sensor as a focus of EPHB4-RASA1 regulation of OSS-induced Ras-MAPK signaling mediated through physical interaction. These findings contribute to an understanding of the mechanism by which EPHB4, RASA1 and Ras regulate lymphatic valvulogenesis.