Previous analysis on the thymus of erythropoietin-producing hepatocyte kinases (Eph) B knockout mice and chimeras revealed that Eph-Eph receptor-interacting proteins (ephrins) are expressed both on T cells and thymic epithelial cells (TECs) and play a role in defining the thymus microenvironments. In the current study, we have used the Cre-LoxP system to selectively delete ephrin-B1 and/or ephrin-B2 in either thymocytes (EfnB1(thy/thy), EfnB2(thy/thy), and EfnB1(thy/thy)EfnB2(thy/thy) mice) or TECs (EfnB1(tec/tec), EfnB2(tec/tec), and EfnB1(tec/tec)EfnB2(tec/tec) mice) and determine the relevance of these Eph ligands in T cell differentiation and thymus histology. Our results indicate that ephrin-B1 and ephrin-B2 expressed on thymocytes play an autonomous role in T cell development and, expressed on TECs, their nonautonomous roles are partially overlapping. The effects of the lack of ephrin-B1 and/or ephrin-B2 on either thymocytes or TECs are more severe and specific on thymic epithelium, contribute to the cell intermingling necessary for thymus organization, and affect cortical TEC subpopulation phenotype and location. Moreover, ephrin-B1 and ephrin-B2 seem to be involved in the temporal appearance of distinct cortical TECs subsets defined by different Ly51 levels of expression on the ontogeny.