The identification of oncogenic cancer driver mutations in combination with the development of targeted cancer therapeutics has represented a major step forward in the treatment of many cancer types. In this study, we aimed to identify and characterize novel therapeutic targets in T-cell acute lymphoblastic leukemia (T-ALL), an aggressive form of leukemia originating from developing T-cells. For this, we focused mainly on protein tyrosine kinases (PTKs), a family of enzymes that is known to regulate the growth of normal cells, and that is strongly implicated in the pathogenesis of cancer.Since activating mutations in the PTK JAK1 were recently identified in T-ALL, we first performed a genetic screen to investigate if these mutations could be detected in commonly used T-ALL cell lines. Although we identified several JAK1 variants, none of these could be confirmed as tumor driver mutations, highlighting the importance of functional validation of variants identified in genetic screens. To identify new PTK therapeutic targets in T-ALL, we then performed functional RNAi screens with a PTK focused RNAi library in 10 human T-ALL cell lines. We could validate RNAi screens as a potent tool for high throughput identification of critical PTK drug targets in leukemia cells and identified several PTK hits that could play a novel uncharacterized role in the pathogenesis of T-ALL. For further hit characterization, we focused on two gene families that are known to play a critical role in normal T-cell development; the JAK and SRC family kinases. In the T-ALL cell line DND-41, we identified a novel activating mutation targeting the IL-7 cytokine receptor, causing JAK1 hyperactivity in these cells. Furthermore, we identified novel inactivating mutations targeting the protein tyrosine phosphatase CD45 (PTPRC), a known negative regulator of SRC and JAK kinase signaling. Using a genetic screen, we validated the presence of CD45 inactivating mutations in T-ALL cell lines and patients and observed that inactivation of CD45 occurred together with activating mutations in IL-7Ralfa, JAK1 or LCK. In functional experiments, loss of CD45 activity enhanced the transforming properties of these oncoproteins and sensitized T-cells to cytokine stimulation, as observed by increased JAK/STAT signaling, whereas overexpression of CD45 decreased cytokine-induced signaling. Taken together, these data indicate a tumor suppressor role for CD45 in T-ALL. In addition, RNAi screening identified a novel critical role for the SRC family kinase LCK in NUP214-ABL1 positive T-ALL and mass spectrometric analysis of NUP214-ABL1 protein complexes led to the identification of three new NUP214-ABL1 interactors, MAD2L1, SMC4 and NUP155, that are strictly required for proliferation and survival of NUP214-ABL1 positive T-ALL cells, hereby providing novel therapeutic targets for the treatment of NUP214-ABL1 positive T-ALL.