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Several in vitro models have been developed to mimic CLL proliferation in immune niches; however, they typically do not induce robust proliferation. We prepared a novel model based on mimicking T-cell signals. Six supportive cell lines were prepared by engineering HS5 stromal cells with stable expression of human CD40L, IL4, IL21, and their combinations. Co-culture with HS5 expressing CD40L and IL4 led to mild CLL cell proliferation (median 7% at day 7), while the HS5 expressing CD40L, IL4, and IL21 led to unprecedented proliferation rate of 44%, which is higher and more reproducible then in other available models. The co-cultures mimicked the gene expression fingerprint of lymph node CLL cells (MYC, NFkB, and E2F signatures; as defined by Herishanu et al, 2011). The other induced pathways reveal novel CLL vulnerabilities in context of CLL-T cell-induced proliferation, and we tested 10 inhibitors based on these data. This revealed for the first time that RAF inhibitors and FOXO1 inhibitors block CLL proliferation. The co-culture model can be downscaled to five microliter volume for large drug screening purposes or upscaled to CLL PDXs by HS5-CD40L-IL4±IL21 co-transplantation. We co-transplanted 41 NSG mice with CLL cells and HS5-CD40L-IL4 or HS5-CD40L-IL4-IL21 using a subcutaneous scaffold and intraperitoneal injection leading to 47% or 82% engraftment efficacy, respectively, with ~20% of PDXs being clonally related to CLL. This shows that CLL cell engraftment in NSG mice can be supported by engineered HS5 cells, thus bypassing the need to use primary T cells in PDX (Bagnara et al, 2011).