T cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy caused by acquisition of genetic alterations during T-cell development. The 5-year overall survival of pediatric T-ALL patients has improved considerably over the past 30 years, largely due to improved risk-based stratification and applying aggressive combination chemotherapies. Classical chemotherapy treatment, however, proves inferior in the treatment of relapsed and refractory T-ALL, demanding the implementation of novel therapeutic strategies. NOTCH1 was identified as one of the most frequently mutated genes in T-ALL and was found often mutated in other cancers. This finding boosted the development of a spectrum of Notch-targeting therapies. These include neutralizing antibodies against Notch receptors and ligands as well as gamma-secretase inhibitors (GSI) that prevent NOTCH receptor cleavage. Previously, we identified and validated a novel orally active small molecule (CB-103) that efficiently blocks the Notch transcription activation complex. Although novel therapeutics blocking Notch signaling show promising outcomes, it is well-known that the use of mono-therapies often results in relapse due to tumor heterogeneity or therapy-induced resistance. Thus, a better understanding of resistance mechanisms to Notch inhibitors and the development of combination therapies will facilitate effective treatment of T-ALL patients. Here, we performed a genome-wide CRISPR-Cas9 screen in human T-ALL cells and identified the Phosphoinositide-3-Kinase regulatory subunit 1 (PIK3R1) as a key player in NOTCH-targeting treatment response. Mutational loss of PIK3R1 activity confers resistance to pharmacological Notch inhibition. Unbiased transcriptomic and proteomic analyses in PIK3R1 deficient T-ALL cells revealed PI3K-AKT mediated up-regulation of pro-survival and proliferation pathways, together with alterations of the spliceosome machinery in response to Notch inhibition. Moreover, we identified and validated a variety of combination therapies, which resulted in reduced tumor burden and prolonged survival in a preclinical xenograft T-ALL model. Overall, our study identified novel resistance mechanisms to pharmacological Notch inhibition and combination strategies capable of more efficient treatment of refractory or relapsed T-ALL patients.