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Mammalian infection by dimorphic fungi occurs through the inhalation of asexual spores (conidia), which are phagocytosed by host pulmonary alveolar macrophages of the innate immune system. Once phagocytosed, fungal conidia germinate into the pathogenic cell type; unicellular yeast cells which divide by fission (Vanittanakom et al., 2006; Boyce et al., 2011). To investigate if mutation of a particular fungal gene affects macrophage phagocytosis or the production of yeast cells, a murine macrophage cell culture assay can be utilized. This protocol was developed for Penicillium marneffei but is applicable to most dimorphic fungi.