Background: Acute lymphoblastic leukaemia with mixed lineage leukaemia gene rearrangement (MLL-ALL) frequently affects infants and is associated with a poor prognosis. Primary refractory and relapsed disease due to resistance to glucocorticoids (GCs) remains a substantial hurdle to improving clinical outcomes. In this study, we aimed to overcome GC resistance of MLL-ALL. Methods: Using leukaemia patient specimens, we performed bioinformatic analyses to identify target genes/pathways. To test inhibition of target pathways in vivo, we created pre-clinical therapeutic mouse patient-derived xenograft (PDX)-models by transplanting human MLL-ALL leukaemia initiating cells (LIC) into immune-deficient NSG mice. Finally, we conducted B-cell lymphoma-2 (BCL-2) homology domain 3 (BH3) profiling to identify BH3 peptides responsible for treatment resistance in MLL-leukaemia. Findings: Src family kinases (SFKs) and Fms-like tyrosine kinase 3 (FLT3) signaling pathway were over-represented in MLL-ALL cells. PDX-models of infant MLL- ALL recapitulated GC-resistance in vivo but RK-20449, an inhibitor of SFKs and FLT3 eliminated human MLL-ALL cells in vivo, overcoming GC-resistance. Further, we identified BCL-2 dependence as a mechanism of treatment resistance in MLL-ALL through BH3 profiling. Furthermore, MLL-ALL cells resistant to RK-20449 treatment were dependent on the anti-apoptotic BCL-2 protein for their survival. Combined inhibition of SFKs/FLT3 by RK-20449 and of BCL-2 by ABT-199 led to substantial elimination of MLL-ALL cells in vitro and in vivo. Triple treatment combining GCs, RK-20449 and ABT-199 resulted in complete elimination of MLL-ALL cells in vivo. Interpretation: SFKs/FLT3 signaling pathways are promising targets for treatment of treatment-resistant MLL-ALL. Combined inhibition of these kinase pathways and anti-apoptotic BCL-2 successfully eliminated highly resistant MLL-ALL and demonstrated a new treatment strategy for treatment-resistant poor-outcome MLL-ALL. Funding: This study was supported by RIKEN (RIKEN President's Discretionary Grant) for FI, Japan Agency for Medical Research and Development (the Basic Science and Platform Technology Program for Innovative Biological Medicine for FI and by NIH CA034196 for LDS. The funders had no role in the study design, data collection, data analysis, interpretation nor writing of the report.