NOTCH1 gain-of-function mutations are recurrent in B-cell chronic lymphocytic leukemia (B-CLL), where they are associated with accelerated disease progression and refractoriness to chemotherapy. The specific role of NOTCH1 in the development and progression of this malignancy is unclear. Here, we assess the impact of loss of Notch signaling and pathway hyperactivation in an in vivo mouse model of CLL (IgH.TE mu) that faithfully replicates many features of the human pathology. Ablation of canonical Notch signaling using conditional gene inactivation of RBP-J in immature hematopoietic or B-cell progenitors delayed CLL induction and reduced incidence of mice developing disease. In contrast, forced expression of a dominant active form of Notch resulted in more animals developing CLL with early disease onset. Comparative analysis of gene expression and epigenetic features of Notch gain-of-function and control CLL cells revealed direct and indirect regulation of cell cycle-associated genes, which led to increased proliferation of Notch gain-of-function CLL cells in vivo. These results demonstrate that Notch signaling facilitates disease initiation and promotes CLL cell proliferation and disease progression.