Dengue virus (DENV) is a significant threat to public health in tropical and subtropical regions, where the frequency of human migration is increasing. Transmission of DENV from donors to recipients after hematopoietic stem cell transplantation has been steadily described. However, the underlying mechanisms remain unclear. Freshly isolated bone marrow (BM) was subjected to DENV infection, followed by multicolor fluorescence-activated cell sorting (FACS) analysis. Virus in supernatants was collected and analyzed by plaque assay. DENV-1 to DENV-4 could effectively infect freshly obtained BM and produced infectious virus. DENV infection did not change the quantitative population of hematopoietic stem and progenitor cells (HSPCs), megakaryocytic progenitor cells (MkPs) and megakaryocytes. Additionally, DENV antigen, nonstructural protein 1, was enriched in HSPCs and MkPs of DENV infected marrow cells. CD34+, CD133+, or CD61+ cells sorted out from BM were not only the major contributing targets facilitating the DENV infection directly but also facilitated the spread of DENV into other cells when cocultured. Results suggest that DENV can efficiently infect HSPCs, which might jeopardize the recipients if DENV-infected cells were subsequently used. We therefore raise the need for DENV screening for both the donors and recipients of hematopoietic stem cell transplantation, especially for donors exposed to endemic areas, to mitigate DENV infection in immunocompromised recipients. © 2019 AABB.