Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of mature CD5 B cells in blood. Spontaneous apoptosis of CLL cells has hampered in-depth investigation of CLL pathogenesis. Here we describe the generation of three monoclonal mouse cell lines, EMC2, EMC4 and EMC6, from the CLL mouse model based on sporadic expression of SV40 large T antigen. The cell lines exhibit a stable CD5CD43IgMCD19 CLL phenotype in culture and can be adoptively transferred into mice. RNA-seq analysis revealed only minor differences between the cell lines and their primary tumors and suggested that NF-κB and mTOR signaling pathways were involved in cell line outgrowth. survival and proliferation was dependent on constitutive phosphorylation of Bruton's tyrosine kinase (Btk) at Y551/Y223, and Akt(S473). Treatment of the cell lines with small molecule inhibitors specific for Btk (ibrutinib) or PI3K (idelalisib), which is upstream of Akt, resulted in reduced viability, proliferation and fibronectin-dependent cell adhesion. Treatment of cell line-engrafted mice with ibrutinib was associated with transient lymphocytosis, reduced splenomegaly and increased overall survival. Thus, by generating stable cell lines we established a novel platform for and investigation of CLL signal transduction and treatment modalities.