Despite substantial progress in treatment of T-cell acute lymphoblastic leukemia, mortality remains relatively high, mainly due to primary or acquired resistance to chemotherapy. Further improvements in survival demand better understanding of T-cell acute lymphoblastic leukemia biology and development of new therapeutic strategies. The Notch pathway has been involved in the pathogenesis of this disease and various therapeutic strategies are currently under development, including selective targeting of Notch receptors by inhibitory antibodies. We previously demonstrated that the Notch1-specific neutralizing antibody OMP52M51 prolongs survival in T-cell acute lymphoblastic leukemia patient-derived xenografts bearing NOTCH1/FBW7 mutations. However, acquired resistance to OMP52M51 eventually developed and we used patient-derived xenografts models to investigate this phenomenon. Multi-level molecular characterization of T-cell acute lymphoblastic leukemia cells resistant to Notch1 blockade and serial transplantation experiments uncovered heterogeneous types of resistance, not previously reported with other Notch inhibitors. In one model, resistance appeared after 156 days of treatment, it was stable and associated with loss of Notch inhibition, reduced mutational load and acquired Notch1 mutations potentially affecting the stability of the heterodimerization domain. Conversely, in another model resistance developed after only 43 days of treatment despite persistent down-regulation of Notch signaling and it was accompanied by modulation of lipid metabolism and reduced surface expression of Notch1. Our findings shed light on heterogeneous mechanisms adopted by the tumor to evade Notch1 blockade and support clinical implementation of antibody-based target therapy for Notch-addicted tumors.