The Notch signaling pathway plays a fundamental role during blood vessel development. Notch signaling regulates blood vessel morphogenesis by promoting arterial endothelial differentiation and providing spatial and temporal control over "tip cell" phenotype during angiogenic sprouting. Components of the Notch signaling pathway have emerged as potential regulators of lymphatic development, joining the increasing examples of blood vessel regulators that are also involved in lymphatic development. However, in mammals a role for the Notch signaling pathway during lymphatic development remains to be demonstrated. In this report, we show that blockade of Notch1 and Dll4, with specific function-blocking antibodies, results in defective postnatal lymphatic development in mice. Mechanistically, Notch1-Dll4 blockade is associated with down-regulation of EphrinB2 expression, been shown to be critically involved in VEGFR3/VEGFC signaling, resulting in reduced lymphangiogenic sprouting. In addition, Notch1-Dll4 blockade leads to compromised expression of distinct lymphatic markers and to dilation of collecting lymphatic vessels with reduced and disorganized mural cell coverage. Finally, Dll4-blockade impairs wound closure and severely affects lymphangiogenesis during the wound healing in adult mouse skin. Thus, our study demonstrates for the first time in a mammalian system that Notch1-Dll4 signaling pathway regulates postnatal lymphatic development and pathologic lymphangiogenesis.