Chronic lymphocytic leukemia (CLL) is a B cell malignancy and represents the most common leukemia in adults within the Western world. This disease mainly affects the elderly, with a median age of diagnosis over 70. The disease course is highly variable among patients; some experience rapidly progressing leukemia which becomes resistant to treatment, while others bear indolent disease and will die from other causes. Morbidity correlates with the chromosomal aberration and mutational burdens observed in patients. NOTCH1 gain-of-function mutations are among the most frequently observed mutations in patients. They are found in 10-15% of CLL patients at diagnosis and their prevalence increases up to 30% in late-stage disease. Many correlative studies have shown that patients harboring NOTCH1 mutations exhibit a reduced survival compared to those with unmutated NOTCH1. Although the presence of NOTCH1 mutations is associated with poor prognosis, it remains unclear by which mechanisms NOTCH signaling might influence disease development. Here, we assessed the role of Notch signaling in CLL using genetic loss- and gain-of-function studies in a transgenic mouse model (IgH.TEµ) that faithfully recapitulates human disease phenotypes. We found that B cell specific knock-out of RBP-J significantly decreased prevalence of CLL induction in RBP-J depleted mice compared to control animals. Reciprocal Notch gain-of-function experiments revealed that mice with enforced Notch1 signaling had an earlier CLL onset compared to controls. Recipients of sorted CLL-like cells over-expressing Notch1 had reduced survival compared to control infusion. Taken together, these genetic studies strongly suggest a role for Notch signaling in disease initiation. To study the molecular mechanisms by which Notch1 signaling enhanced CLL aggressiveness, global gene expression as well as global epigenetic analyses were performed on sorted CLL-like cells from Notch1 gain-of-function and control animals. Cell cycle related pathways in gene set enrichment analysis were the most significantly induced signature in the Notch1 gain-of function CLL-like cells compared to control CLL-like cells. This result was further confirmed by functional cell cycle assays in vivo, strongly suggesting that Notch signaling accelerates CLL initiation by promoting proliferation.