The pre-T cell receptor (TCR) functions as a critical checkpoint during αβ T cell development. Signaling through the pre-TCR controls the differentiation of immature CD4^-CD8^-CD25⁺CD44^- [double-negative (DN)3] thymocytes into CD4⁺CD8⁺ double-positive (DP) cells through the CD4^-CD8^-CD25^-CD44^-(DN4) stage. In addition, pre-TCR activity triggers expansion and survival of thymocytes and inhibits TCRβ gene rearrangement through a process referred to as allelic exclusion. Whereas many proteins involved in the pre-TCR transduction cascade have been identified, little is known about the nuclear factors associated with receptor function. Here, we use gene targeting to inactivate the Ets-1 transcription factor in mice and analyze pre-TCR function in developing Ets-1-deficient (Ets-1^-/-) thymocytes. We find that inactivation of Ets-1 impairs the development of DN3 into DP thymocytes and induces an elevated rate of cell death in the DN4 subset. This defect appears specific to the αβ lineage because γδ T cells maturate efficiently. Finally, the percentage of thymocytes coexpressing two different TCRβ chains is increased in the Ets-1^-/- background and, in contrast with wild type, forced activation of pre-TCR signaling does not block endogenous TCRβ gene rearrangement. These data identify Ets-1 as a critical transcription factor for pre-TCR functioning and for allelic exclusion at the TCRβ locus.