Hematopoietic stem/progenitor cell (HSPC)-based anti-HIV-1 gene therapy holds promise to provide life-long remission following a single treatment. Here we report a multi-pronged anti-HIV-1 HSPC-based gene therapy designed to defend against and attack HIV-1 infection. We developed a lentiviral vector capable of co-expressing three anti-HIV-1 genes. Two are designed to prevent infection, including a short-hairpin RNA (CCR5sh1005) to knock down HIV-1 co-receptor CCR5 and a membrane anchored HIV-1 fusion inhibitor (C46). The third gene is a CD4-based chimeric antigen receptor (CAR) designed to attack HIV-1 infected cells. Our vector also includes a non-signaling truncated human epidermal growth factor receptor (huEGFRt) which acts as a negative selection-based safety kill switch against transduced cells. Anti-HIV-1 vector-transduced human CD34+ HSPC efficiently reconstituted multi-lineage human hematopoietic cells in humanized bone marrow/liver/thymus (huBLT) mice. HIV-1 viral load was significantly reduced (1-log fold reduction, p <0.001) in transplanted huBLT mice. Anti-huEGFR monoclonal antibody Cetuximab (CTX) administration significantly reduced huEGFRt+ vector-modified cells (>4-fold reduction, p <0.01) in huBLT mice. These results demonstrate that our strategy is highly effective for HIV-1 inhibition, and that CTX-mediated negative selection can deplete anti-HIV-1 vector-modified cells in the event of unwanted adverse effects in huBLT mice.