Human immunodeficiency virus-1-derived lentiviral vectors have been increasingly used for gene delivery in both pre-clinical and clinical models. Numerous studies have shown that dendritic cells (DC) transduced with concentrated lentiviral vectors can induce primary T-cell responses to viral and tumor antigens. In this study, we attempted to generate influenza hemagglutinin-specific CD4 T cells using lentiviral vectors containing the signal sequence and human lysosome-associated membrane protein to target hemagglutinin to the major histocompatibility complex class II processing pathway. Autologous dendritic cells were generated in serum-free medium and transduced with concentrated, high-titer lentiviruses to stimulate autologous T cells. Unexpectedly, we failed to generate influenza hemagglutinin-specific CD4 T cells rather than T cells specific for fetal calf serum (FCS). By limiting dilution, we established several FCS-specific CD4 T-cell clones restricted by human leukocyte antigen-DR1 and human leukocyte antigen-DR4. Lentiviruses produced in human serum-adapted 293 cells or in serum-free medium were unable to sensitize dendritic cells for recognition by FCS-specific CD4 T-cell clones. Our results indicate that residual FCS in concentrated lentiviral pellets is, in part, responsible for its immunogenicity. These FCS-specific CD4 T cells may be useful in testing clinical grade lentiviral vectors for the presence of contaminating FCS.