HIV-1 impairs the production of T cells, through mechanisms that are still unknown. Here, we investigated the effect of the expression of HIV-1 Nef on the T-cell potential of human hematopoietic CD34 + precursors. Those progenitors were transduced by using lentiviral vectors expressing Nef and cultured on OP9-DL1 cells allowing the differentiation of T cell from human hematopoietic precursors. We demonstrate that Nef impairs the generation of a CD3ɛ + CD5 + CD1a + precursor stage that has initiated a D-J rearrangement of the TCRβ locus.Onward stages of T-cell development were also affected with a quantitative reduction of CD4 + intraCD3ɛ + Immature single positive cells (ISP), Double Positive (DP) CD4 + CD8 + TCRαβ T cells and CD56 + NK cells. But B cell production was not affected. Limiting dilution analyses demonstrated a significant reduction in the frequency of T/NK progenitors among Nef-expressing CD34 + cells. Altogether, these data demonstrate that Nef interferes with the differentiation of a primitive lymphoid human precursor with a T/NK potential.