We have previously suggested that asymmetric cell division might represent a mechanism to ensure that appropriate diversity of cell fate arises from the descendants of a single lymphocyte during an immune response. We now show that the fate-determining transcription factor T-bet is asymmetrically inherited by dividing CD4+ and CD8+ T cells recruited into an immune response. T-bet is induced in interphase T cells within hours of activation. During mitosis, T-bet undergoes proteasome-dependent degradation. Mitotic destruction is mediated by T cell receptor-induced tyrosine phosphorylation of T-bet. Unequal inheritance of T-bet is associated with asymmetric segregation of the proteasomal degradative machinery during mitosis and cytokinesis. Mutations of T-bet at the critical tyrosine and those disabling the T cell receptor-associated kinase, ITK, both result in symmetric inheritance of T-bet without affecting asymmetry of the proteasome. These results suggest that two experimentally distinct mechanisms promote the unequal inheritance of T-bet by initial daughter T cells: one signal that targets T-bet for mitotic destruction and another signal that renders inequality in the inheritance of the cellular machinery that destroys T-bet. These findings offer a new framework for understanding how signaling to a single T lymphocyte can result in unequal fate determination of its daughter cells.