HIV infection has been associated with defective hematopoiesis since the earliest days of the HIV/AIDS epidemic. Generation of all hematopoietic lineages suffers in the face of infection. The mechanisms by which HIV impairs normal blood cell development remain unclear, and direct infection of intermediate hematopoietic progenitors has not been established as a source of HIV-associated hematopoietic pathology. Here, we demonstrate infection of multiple subsets of highly purified intermediate hematopoietic progenitors by wild-type HIV both in vitro and in vivo. Although direct infection is clearly cytotoxic, we find that some infected progenitors can survive and harbor proviral DNA. We report intermediate hematopoietic progenitors to be a novel target of infection and their permissivity to infection increases with development. Further, the nonobese diabetic severe combined immunodeficiency common γ chain knockout-bone marrow-liver-thymus humanized mouse provides a unique model for studying the impact of HIV infection on bone marrow–based human hematopoiesis.