Notch proteins influence cell fate decisions in many developmental systems. During lymphoid development, Notch1 signaling is essential to direct a bipotent T/B precursor toward the T cell fate, but the role of Notch1 at later stages of T cell development remains controversial. We have recently reported that tissue-specific inactivation of Notch1 in immature (CD44⁻ CD25⁺) thymocytes does not affect subsequent T cell development. Here, we demonstrate that loss of Notch1 signaling at an earlier (CD44⁺CD25⁺) developmental stage results in severe perturbation of αβ but not γδ lineage development. Immature Notch1^−/− thymocytes show impaired VDJβ rearrangement and aberrant pre-TCR-independent survival. Collectively, our data demonstrate that Notch1 controls several nonredundant functions necessary for αβ lineage development.