Abstract: Elucidating the exact contribution of microglia to central nervous system (CNS) pathology has historically been extremely challenging. These resident parenchymal myeloid cells are considered to have critical roles as frontline responders during pathogen invasion and CNS perturbation. Thus, understanding the precise temporal kinetics of microglial function is central to the evolution of novel therapeutics for disease intervention and/or resolution (Spiteri et al., 2022a). The development of PLX5622, a colony-stimulating factor 1 receptor (CSF-1R) inhibitor typically formulated into a rodent chow for simple oral administration has facilitated exploration of microglial functions in disease (Spangenberg et al., 2019). This molecule is widely used as a microglia-depletion agent, with a 20-fold greater selectivity for CSF-1R than other kinases, greater CNS penetrance and better depletion efficacy than previously developed CSF-1R inhibitors like PLX3397. Moreover, PLX5622 is more effective than other microglia depletion systems, including those using clodronate depletion, or CD11b-HSVTK mice that express the herpes-simplex virus thymidine kinase under the CD11b-promoter (Heppner et al., 2005), or CX3CR1CreER: iDTR mice that express Cre-recombinase under the CX3CR1 promoter and crossed with iDTR animals (Bruttger et al., 2015). These approaches require intracranial injections, bone marrow (BM)-reconstitutions, or administration of ganciclovir or tamoxifen, all of which produce non-physiological effects that may confound data interpretation. However, while PLX5622 has undoubtedly enhanced our understanding of microglia biology, studies investigating the potential off-target or indirect effects of this molecule are few and have focused on circulating blood leukocytes or splenocytes. Moreover, these studies have been confined to major cell subsets due to the limited array of cytometric parameters used, and thus the identification of smaller, equally important immune subsets have not been reported. More recently, ex vivo analysis showed impairment in macrophages and lymphoid subsets 3 weeks post-PLX5622 treatment (Lei et al., 2020). Elucidating the precise impact of this molecule on the periphery is important, particularly if PLX5622 is used to study the role of microglia in diseases where peripheral immune cells demonstrably contribute to disease resolution and/or progression, as seen in viral encephalitis. Indeed, modulation of peripheral immune cell compartments by indirect effects of PLX5622 substantially impeded dissection of the role of microglia in a murine model of West Nile virus (WNV) encephalitis (WNE) (Spiteri et al., 2022b; Figure 1).