Macrophages (Mϕ) are the major source of inflammatory cytokines and are target cells for dengue virus (DV) replication. However, Mϕ are heterogeneous and their phenotypic and functional diversities are influenced by cytokines that regulate their differentiation, tissue distribution, and defense against invading pathogens. In vitro, human primary macrophages are derived from peripheral blood CD14+ monocytes in the presence of macrophage colony-stimulating factor (M-CSF) or granulocyte macrophage colony-stimulating factor (GM-CSF). These are essential for developing tissue/resting macrophages (M-Mϕ) and inflammatory macrophages (GM-Mϕ), respectively. While IFN production is similar between M-Mϕ and GM-Mϕ, M-Mϕ cannot produce IL-1β after DV infection. In contrast, GM-Mϕ is more susceptible to DV infection and DV triggers CLEC5A in GM-Mϕ to activate NLRP3 inflammasomes, which in turn release IL-18 and IL-1β that are critical for Th17 activation and contribute to disease severity. Thus, GM-Mϕ is more representative than M-Mϕ for investigating inflammasome activation in dengue infection, and is invaluable for revealing the molecular mechanism of pathogen-induced inflammatory reaction. Distinct phenotypes of macrophage subsets under the influence of M-CSF and GM-CSF raise the question of optimal conditions for culturing primary macrophages to study host-pathogen interaction.