TCR gene rearrangement and expression are central to the development of clonal T lymphocytes. The pre-TCR complex provides the first signal instructing differentiation and proliferation events during the transition from CD4− CD8− TCR− double negative (DN) stage to CD4+ CD8+ double positive (DP) stage. How the pre-TCR signal leads to downstream gene expression is not known. HeLa E-box binding protein (HEB), a basic helix-loop-helix transcription factor, is abundantly detected in thymocytes and is thought to regulate E-box sites present in many T cell-specific gene enhancers, including TCR-α, TCR-β, and CD4. Targeted disruption of HEB results in a 5-to 10-fold reduction in thymic cellularity that can be accounted for by a developmental block at the DN to DP stage transition. Specifically, a dramatic increase in the CD4 low/− CD8+ CD5 low HSA+ TCR low/− immature single positive population and a concomitant decrease in the subsequent DP population are observed. Adoptive transfer test shows that this defect is cell-autonomous and restricted to the αβ T cell lineage. Introduction of an αβ TCR transgene into the HEB ko/ko background is not sufficient to rescue the developmental delay. In vivo CD3 cross-linking analysis of thymocytes indicates that TCR signaling pathway in the HEB ko/ko mice appears intact. These findings suggest an essential function of HEB in early T cell development, downstream or parallel to the pre-TCR signaling pathway.