PBX1 is a proto-oncogene that plays important roles in pattern formation during development. It was discovered as a fusion with the E2A gene after chromosomal translocations in a subset of acute leukemias. The resulting E2a-Pbx1 chimeric proteins display potent oncogenic properties that appear to require dimerization with Hox DNA binding partners. To define molecular pathways that may be impacted by E2a-Pbx1, a genetic screen consisting of neonatal retroviral infection was used to identify genes that accelerate development of T-cell tumors in E2A-PBX1 transgenic mice. Retroviral insertions in the Notch1 gene were observed in 88% of tumors arising with a shortened latency. Among these, approximately half created a Notch^IC allele, encoding the intracellular, signaling portion of Notch1, suggesting a synergistic interaction between the Notch and E2a-Pbx1 pathways in oncogenesis. The remaining proviral insertions involvingNotch1 occurred in a more 3′ exon, resulting in truncating mutations that deleted the carboxy-terminal region ofNotch1 containing negative regulatory sequences (Notch1^ΔC). In contrast toNotch^IC, forced expression ofNotch1^ΔC in transgenic mice did not perturb thymocyte growth or differentiation. However, mice transgenic for both the E2A-PBX1 and Notch1^ΔC genes displayed a substantially shortened latency for tumor development compared with E2A-PBX1 single transgenic mice. These studies reveal a novel mechanism for oncogenic activation ofNotch1 and demonstrate a collaborative relationship between 2 cellular oncogenes that also contribute to cell fate determination during embryonic development.