The most common current models for anti-HIV-1 gene therapy is through the use of humanized mice, immunodeficient animals into which human cells are engrafted. Evaluation of anti-HIV-1 gene therapy in animal models has been performed by HIV-1 challenge AFTER transplant with gene modified HSPC. This experimental design allows us to gain important data in a relatively simple setting. However, the clinical application of gene therapy will be in patients already infected with HIV-1. Thus, it is important to test the impact of HIV-1 infection on the success of gene modified HSPC engraftment and differentiation and through control of HIV-1 in previously infected humanized mice. We tested a new mouse model whereby the first transplant of CD34+ cells allows human hematopoietic cell reconstitution and provides human cells for HIV-1. After HIV-1 is established, a second transplant is conducted using anti-HIV-1 gene modified CD34+ cells and thymus tissue. We find that the timing of the second transplant is important in order to maintain the integrity of the thymic organoid. In this system, repopulation of human CD45+ cells, CD3+, CD4+, CD8+ cells occurs efficiently. In HIV-1 infected animals, CD4+ cells are decreased relative to uninfected control animals. Under these conditions, we observe protection from HIV-1 by the transplant of genetically modified CD34+ cells. These studies provide the basis for a new model which should be useful for understanding and evaluating the efficacy of anti-HIV-1 gene modification of hematopoietic stem cells.