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Our understanding about medullary compartment, its niches composition and formation is still limited. Previous studies using EphB2 and/or EphB3 knockout mice showed an abnormal thymic development that affects mainly to the epithelial component, including the cortex/medulla distribution, thymic epithelial cell (TEC) morphology and different epithelial-specific marker expression. We have already demonstrated that the lack of ephrinB1 and/or ephrinB2, either on thymocytes or on TECs, alters the cell intermingling processes necessary for thymus organization and affect cortical TEC subpopulations. In the present work, we have used the Cre–LoxP model to selectively delete ephrinB1 and/or ephrinB2 in thymocytes (EfnB1thy/thy, EfnB2thy/thy, EfnB1thy/thyEfnB2thy/thy mice) or TECs (EfnB1tec/tec, EfnB2tec/tec, EfnB1tec/tecEfnB2tec/tec mice) and have analyzed their role on the medullary compartment. In all the studied mutants, medullary areas are smaller and more compact than in the wt thymuses. In most of them, we observe abundant big cysts and a higher proportion of UEAhiMTS10− cells than in wt mice, which are often forming small cysts. On EfnB1tec/tecEfnB2tec/tec, changes affecting organ size and medullary compartment start at perinatal stage. Our data shed some light on knowledge about wt medulla histological structure and cysts meaning and formation process and on the role played by ephrinB in them., Ministerio de Educación y Ciencia (España), Ministerio de Sanidad y Consumo (España), Gobierno Regional de Madrid (España), Depto. de Biología Celular, Fac. de Ciencias Biológicas, TRUE, pub