SUMMARY Notch signaling is a fundamental regulator of lymphatic growth, function and vessel homeostasis. Lymphatic dysfunction is associated with many acquired human diseases, including liver fibrosis, psoriasis, metabolic diseases, and cancer. The postnatal lymphatic endothelium dynamically expresses both Notch1 and Notch4, as well as the Notch ligands, Jag1 and Dll4. Numerous therapies that target Notch pathway components are in clinical development or trials. Thus, it is essential to understand the role of the distinct receptors and ligands of the Notch pathway in the adult lymphatic vasculature. Downstream of Dll4- and Jag1-activation, we found that Notch1 and Notch4 have overlapping and distinct transcriptional profiles in lymphatic endothelial cells (LECs), suggesting a dynamic role for Notch in LECs. Our studies of Notch4-/- mice indicate that Notch4 regulates dermal lymphatic vascular patterning, while it is necessary specifically in adult females for the regeneration and maintenance of dermal lymphatics. Using in vitro studies, we show that Notch4 regulates proliferation, and VECADHERIN expression posttranslationally, which in turn may affect LEC growth, responses to flow and barrier function. Postnatal deletion of LEC Notch4 using a novel transgenic model led to reduced dermal capillary lymphatics and LEC proliferation and altered VECADHERIN expression. Analysis of male N4LECKO mice revealed increased hepatic portal lymphatics associated with multiple hallmarks of pathological liver disease and fibrosis. We hypothesize that Dll4 and Jag1 dynamically signal via Notch1 and Notch4 in capillary lymphatics to maintain lymphatic vessel homeostasis and regeneration in the adult. To address these hypotheses, we propose to 1) transcriptionally profile lymphatic endothelial Notch1 and Notch4 signaling downstream of Dll4 and Jag1 activation using in vitro and in vivo approaches, 2) determine the mechanisms by which Notch4 functions in the dermal lymphatic endothelium of the adult mice, and 3) determine the role of lymphatic endothelial Notch4 in the liver and a mouse model of dietary-induced non-alcoholic steatohepatitis (NASH). The goal is to understand LEC Notch4 functions in lymphatic vascular maintenance and homeostasis and how its loss contributes to pathogenic phenotypes in the skin and liver.