Differentiation of genetically modified CD34⁺ hematopoietic stem cells into dendritic cells (DCs) will contribute to the development of immunotherapeutic anticancer protocols. Retroviral vectors that have been used for the transduction of CD34⁺ cells face the problem of gene silencing when integrated into the genome of repopulating stem cells. We reasoned that a high copy number of retroviral DNA sequences might overcome silencing of transgene expression during expansion and differentiation of progenitor cells into functional DCs. To prove this, we utilized a retroviral vector with bicistronic expression of the melanoma-associated antigen tyrosinase and the enhanced green fluorescent protein (EGFP). Human cord blood CD34⁺ cells were transduced with vesicular stomatitis virus G-protein (VSV-G) pseudotyped Moloney murine leukemia virus (MoMuLV) particles using 100-150 multiplicity of infection. During expansion of transduced cells with immature phenotype, transgene expression was strongly silenced, but upon differentiation into mature DCs, residual transgene expression was retained. Intracellular processing of the provirally expressed tyrosinase was tested in a chromium release assay utilizing a cytotoxic T cell clone specific for a HLA-A*0201-restricted tyrosinase peptide. We suggest that retroviral transduction of tumor-associated antigens in hematopoietic progenitor cells and subsequent differentiation into DCs is a suitable basis for the development of potent anti-tumor vaccines.