Persistent induction of type 1 interferon (IFN) is associated with human immunodeficiency virus type 1 (HIV-1) infection. We report here that the pathogenic HIV strain R3A (HIV-R3A) induced high levels of type 1 IFN, while the nonpathogenic HIV-R3B showed no significant induction in human fetal thymus organ culture (HFTOC). We found that IFN contributed to the depletion of human T cells by HIV-R3A in a fusion-independent fashion. The R3B recombinant with the R3A Env V1V2 domain (R3B/A-V1V2) was able to induce type 1 IFN, which contributed to the increased depletion of T cells. Therefore, type 1 IFN induction plays a significant role in HIV-induced T cell depletion in the human thymus.