The double-positive (DP) to single-positive (SP) transition during T cell development is initiated by downregulation of the E protein transcription factors HEB and E2A. Here, we have demonstrated that in addition to regulating the onset of this transition, HEB and E2A also play a separate role in CD4+ lineage choice. Deletion of HEB and E2A in DP thymocytes specifically blocked the development of CD4+ lineage T cells. Furthermore, deletion of the E protein inhibitors Id2 and Id3 allowed CD4+ T cell development but blocked CD8+ lineage development. Analysis of the CD4+ lineage transcriptional regulators ThPOK and Gata3 placed HEB and E2A upstream of CD4+ lineage specification. These studies identify an important role for E proteins in the activation of CD4+ lineage differentiation as thymocytes undergo the DP to SP transition.