AbstractNumb asymmetrically segregates at mitosis to control cell fate choices during development. Numb inheritance specifies progenitor over differentiated cell fates, and, paradoxically, also promotes neuronal differentiation, thus indicating that the role of Numb may change during development. Here we report that Numb nuclear localization is restricted to early thymocyte precursors, whereas timed appearance of pre-T-cell receptor (pre-TCR) and activation of protein kinase Cθ promote phosphorylation-dependent Numb nuclear exclusion. Notably, nuclear localization of Numb in early thymocyte precursors favors p53 nuclear stabilization, whereas pre-TCR-dependent Numb nuclear exclusion promotes the p53 downmodulation essential for further differentiation. Accordingly, the persistence of Numb in the nucleus impairs the differentiation and promotes precursor cell death. This study reveals a novel regulatory mechanism for Numb function based on its nucleus–cytosol shuttling, coupling the different roles of Numb with different stages of T-cell development.