Studying chronic lymphocytic leukemia (CLL) in vitro is challenging due to its complexity and dependency of CLL cells on their microenvironment. An integral part of the natural CLL microenvironment is the three-dimensional (3D) spatial organization, which facilitates frequent cell-to-cell and cell-to-matrix contacts. Therefore, we aimed to mimic the natural tissue architecture by implementing a 3D in vitro culture. We hypothesized that compared to conventional culture, the additional dimension and increased cell-to-matrix contacts would enhance the prosurvival stimuli in CLL cells. We cultured CLL cells in two materials: collagen scaffolds, or gel composed of carboxymethyl cellulose and polyethylene glycol (CMC-PEG). We assessed cell distribution, morphology, and viability via microscopy, and measured the metabolic activity by AlamarBlue assay. Gene expression (MYC, VCAM1, MCL1, CXCR4, CCL4) was analyzed using qPCR to understand the effects of novel culture approaches on adhesion, apoptosis, and intercellular interactions of CLL cells co-cultured with bone marrow stromal cells (BMSCs) in 3D.