Notch signaling regulates lineage decisions at multiple stages of lymphocyte development, and Notch activation requires the endocytosis of Notch ligands in the signal-sending cells. Four E3 ubiquitin ligases, Mind bomb (Mib) 1, Mib2, Neuralized (Neur) 1, and Neur2, regulate the Notch ligands to activate Notch signaling, but their roles in lymphocyte development have not been defined. We show that Mib1 regulates T and marginal zone B (MZB) cell development in the lymphopoietic niches. Inactivation of the Mib1 gene, but not the other E3 ligases, Mib2, Neur1, and Neur2, abrogated T and MZB cell development. Reciprocal bone marrow (BM) transplantation experiments revealed that Mib1 in the thymic and splenic niches is essential for T and MZB cell development. Interestingly, when BM cells from transgenic Notch reporter mice were transplanted into Mib1-null mice, the Notch signaling was abolished in the double-negative thymocytes. In addition, the endocytosis of Dll1 was impaired in the Mib1-null microenvironment. Moreover, the block in T cell development and the failure of Dll1 endocytosis were also observed in coculture system by Mib1 knockdown. Our study reveals that Mib1 is the essential E3 ligase in T and MZB cell development, through the regulation of Notch ligands in the thymic and splenic microenvironments.