Gain-of-function mutations in NOTCH1 are among the most frequent genetic alterations in T-cell acute lymphoblastic leukemia (T-ALL), highlighting the Notch signaling pathway as a promising therapeutic target for personalized medicine. Yet, a major limitation for long-term success of targeted therapy is relapse due to tumor heterogeneity or acquired resistance. Thus, we performed a genome-wide CRISPR-Cas9 screen to identify prospective resistance mechanisms to pharmacological NOTCH inhibitors and novel targeted combination therapies to efficiently combat T-ALL. Mutational loss of phosphoinositide-3-kinase regulatory subunit 1 (PIK3R1) causes resistance to Notch inhibition. PIK3R1 deficiency leads to increased PI3K/AKT signaling, which regulates cell cycle and the spliceosome machinery, both at the transcriptional and posttranslational level. Moreover, several therapeutic combinations have been identified, in which simultaneous targeting of the cyclin-dependent kinases 4 and 6 (CDK4/6) and NOTCH proved to be the most efficacious in T-ALL xenotransplantation models.