Mutations resulting in overexpression of intracellular Notch1 (ICN1) are frequently observed in human T cell acute lymphoblastic leukemia (T-ALL). We have determined the consequences of ICN1 overexpression from retroviral vectors introduced into bone marrow cells. Early consequences are the generation of polyclonal nontumorigenic CD4⁺8⁺ T cell receptor (TCR)-αβ⁺ cells that do not qualify as tumor precursors despite the observation that they overexpress Notch 1 and c-Myc and degrade the tumor suppressor E2A by posttranslational modification. The first tumorigenic cells are detected among more immature CD4⁻8⁺TCR-αβ⁻ cells that give rise to monoclonal tumors with a single, unique TCR-β chain and diverse TCR-α chains, pinpointing malignant transformation to a stage after pre-TCR signaling and before completion of TCR-α rearrangement. In T-ALL, E2A deficiency is accompanied by further transcriptional up-regulation of c-Myc and concomitant dysregulation of the c-Myc-p53 axis at the transcriptional level. Even though the tumors consist of phenotypically heterogeneous cells, no evidence for tumor stem cells was found. As judged by array-based comparative genomic hybridization (array CGH) and spectral karyotype (SKY) analysis, none of the tumors arise because of genomic instability.