The development and maintenance of thymic microenvironments depends on sustained crosstalk signals derived from developing thymocytes. However, the molecular basis for the initial phase in the lymphoid dependent development of thymic epithelial cells (TECs) remains unclear. Here we show that similarly to regular thymocytes, developing B cells enforced to express the Notch ligand Delta-like-1 (DLL1) efficiently induce the non-polarized, three-dimensional (3D) meshwork architecture of cortical TECs in fetal thymic organ culture. Moreover, the DLL1-overexpressing B cells induce well-developed distinct medullae. Such medullae also arose in lobes reconstituted with Rag2^−/− thymocytes overexpressing DLL1. Our present findings thus strongly suggest that Notch signaling from thymocytes to TECs induces TEC development at an early phase of thymic organogenesis. The present approach using non-T lineage cells for the in vitro construction of thymic environments may also provide a novel tool for thymus regeneration and T cell production in immunocompromised individuals.