Despite recent advances, chronic lymphocytic leukemia (CLL) as the most common leukemia remains a largely incurable disease. Modern treatment options include novel drugs like purine analogues, monoclonal antibodies and transplantation strategies. Moreover, gene transfer of immunostimulatory molecules is another, but still experimental approach that can be used to potentiate immune responses against leukemic cells. CD40 ligand (CD40L) was shown to be a promising molecule for immunotherapy of B-CLL playing a critical role in immune activation. However, CLL B cells are resistant to transduction with most currently available vector systems. Improving the efficiency and specificity of gene vectors is critical for the success of gene therapy in this area. Using replication defective adenovirus encoding CD40L (Ad-CD40L), immunologic and clinical responses were seen in CLL patients after infusion of autologous Ad-CD40L-CLL cells in a recent phase I trial. Due to the immunogenic nature of adenovirus vectors, alternative vector systems are currently explored. Recombinant adeno-associated virus (rAAV) was shown to enable efficient transduction of primary B-CLL cells. By use of a library of AAV clones with randomly modified capsids, receptor-targeting mutants with a tropism for CLL cells can be selected. Furthermore, helper-virus free Epstein-Barr virus (EBV)-based gene transfer vectors hold promise for development of CLL-targeted vaccines after remaining safety issues will be resolved. Herpes simplex virus (HSV)-based vectors, especially HSV amplicons, have favorable features for B-CLL gene transfer including high transduction efficiency, ability to infect postmitotic cells and a large packaging capacity. The challenge for the future will be to transfer these alternative vector systems into clinic and allow the detection of a CLL-specific immune response by use of defined tumor antigens. This will make it possible to establish the potential clinical role of gene therapy for CLL patients.