During development, lymphatic vasculature forms as a second and distinct vascular network derived from embryonic blood vessels. Trans-differentiation of venous endothelial cells into specified lymphatic endothelial cells (LECs) is the first step in this process. Trans-differentiation and specification of LEC fate requires Prox1, but how Prox1 regulates trans-differentiation and maintains lymphatic fate is not fully understood. We present a single cell transcriptomic atlas of lymphangiogenesis in zebrafish that spans four key developmental stages and reveals the highly conserved molecular regulation of lymphatic development in vertebrates. We extend this atlas of wild-type gene expression to comprehensively profile single cell transcriptomic and chromatin changes controlled by Prox1 during the maintenance of lymphatic identity in mutant embryos. This reveals previously unappreciated regulatory relationships for Prox1 in maintenance of chromatin state, in repressing haematopoietic and blood vascular fate and in promoting lymphatic identity. Extending this analysis of cell fate maintenance, we performed single cell analysis of the earliest stages in venous-lymphatic endothelial cell transdifferentiation using prox1a/prox1b double maternal zygotic mutants. This work revealed discrete and dynamic roles for Prox1 at different stages of LEC fate specification and differentiation. Prox1 plays dual and progressive roles, initially blocking blood vascular and hematopoietic fate while simultaneously up-regulating a small number of early acting, functionally essential regulators of lymphatic development including tspan18a/b. This embryonic developmental resource will serve as a baseline to better understand both developmental and pathological lymphangiogenesis in the future.