Targeting Notch signaling has emerged as a promising therapeutic strategy for chronic lymphocytic leukemia (CLL), especially for the poor prognostic subgroup of NOTCH1-mutated patients. Here, we report that the γ-secretase inhibitor PF-03084014 inhibits the constitutive Notch activation and induces selective apoptosis in CLL cells carrying NOTCH1 mutations. Combination of PF-03084014 with fludarabine has a synergistic antileukemic effect in primary NOTCH1-mutated CLL cells, even in the presence of the protective stroma. At transcriptional level, PF-03084014 plus fludarabine treatment induces the upregulation of the proapoptotic gene HRK and the downmodulation of MMP9, IL32 and RAC2 genes that are related to invasion and chemotaxis. PF-03084014 also overcomes fludarabine-mediated activation of nuclear factor-κB signaling. Moreover, this combination impairs angiogenesis and CXCL12-induced responses in NOTCH1-mutated CLL cells, in particular those related to tumoral migration and invasion. Importantly, all these collaborative effects are specific for NOTCH1 mutation and do not occur in unmutated cases. In conclusion, we provide evidence that Notch is a therapeutic target in CLL cases with NOTCH1-activating mutations, supporting the use of Notch pathway inhibitors in combination with chemotherapy as a promising approach for the treatment of these high-risk CLL patients.