The highly pathogenic avian influenza (HPAI) H5N1 virus causes severe systemic infection in avian and mammalian species, including humans by first targeting immune cells. This subsequently renders the innate and adaptive immune responses less active, thus allowing dissemination of the virus to systemic organs. To gain insight into the pathogenesis of H5N1, this study aims to determine the susceptibility of human PBMCs to the H5N1 virus and explore the factors which influence this susceptibility. We found that PBMCs were a target of H5N1 infection, and that monocytes and B cells were populations which were clearly the most susceptible. Analysis of PBMC subpopulations showed that isolated monocytes and monocytes residing in whole PBMCs had comparable percentages of infection (28.97 ± 5.54% vs 22.23 ± 5.14%). In contrast, isolated B cells were infected to a much lower degree than B cells residing in a mixture of whole PBMCs (0.88 ± 0.34% vs 34.87 ± 4.63%). Different susceptibility levels of B cells for these tested conditions spurred us to explore the B cell-H5N1 interaction mechanisms. Here, we first demonstrated that monocytes play a crucial role in the enhancement of B cell susceptibility to H5N1 infection. Although the actual mechanism by which this enhancement occurs remains in question, α2,3-linked sialic acid (SA), known for influenza virus receptors, could be a responsible factor for the greater susceptibility of B cells, as it was highly expressed on the surface of B cells upon H5N1 infection of B cell/monocyte co-cultures. Our findings reveal some of the factors involved with the permissiveness of human immune cells to H5N1 virus and provide a better understanding of the tropism of H5N1 in immune cells