We haver evaluated the regulatory role of mouse Dot1l, a histone H3 lysine (K) 79 (H3K79) methyltransferase, in lymphatic formation. Our studies reveal that Dot1l functions in c-Kit(+) hemogenic ECs during mesenteric lymphatic formation and hence successfully establish that Dot1l-mediated epigenetic priming and transcriptional regulation in LEC progenitors safeguard the proper lymphatic development and functioning of lymphatic vessels.