Oligodendrocytes are the myelinating cells of the central nervous system. Critical to signal conduction via ensheathment of axons, their loss in inflammatory demyelinating diseases such as multiple sclerosis (MS) (Reich et al., 2018) leads to axonal damage and neurodegeneration. Cortical demyelination is a critical contributor to progressive disease in MS. However, the mechanisms underlying and differentiating gray matter demyelination from white matter demyelination, and subsequent remyelination, are not yet fully understood. Pathological studies indicate that gray matter demyelination can occur early in people living with MS and significantly contribute to cognitive deficits (Calabrese et al., 2009; Lucchinetti et al., 1996; Mike et al., 2011; Roosendaal et al., 2009). However, these pathology studies are limited to single time-points, and gray matter lesion detection is limited on clinical MRI (Lucchinetti et al., 2011), so our understanding of the timing of formation, correlation to symptoms and progression, and remyelination potential of cortical lesions is limited. To overcome these limitations we previously combined the cuprizone model to study oligodendrocyte loss and replacement dynamics in the cortex (Orthmann-Murphy et al., 2020). By taking advantage of the precise temporal and spatial resolution of longitudinal two-photon imaging, we found that oligodendrocyte regeneration (and therefore, remyelination) is impaired in deeper layers of the cortex. In this thesis, I adapted the time course of cortical demyelination and recovery defined by in vivo imaging, to test whether microglia are barriers to cortical remyelination. I use a combination of in situ RNA labeling and immunofluorescence to characterize how cortical microglia respond to cuprizone-mediated demyelination and then influence oligodendrocyte regeneration. I show that superficial and deep cortical microglia react to demyelination in distinct ways: deep cortical microglia upregulate CD68, and downregulate homeostatic markers P2RY12 and TMEM119, and adopt an amoeboid morphology characteristic of microglia with high phagocytic and migratory activity (Vidal-Itriago et al., 2022), whereas superficial microglia maintain expression of P2RY12 and TMEM119 as well as a homeostatic morphology (rounded cell body with ramified processes). These spatially- restricted reactive changes persist through two weeks of early recovery post-cuprizone treatment and resolve by 5 weeks of recovery. To determine whether deep cortical reactive microglia directly alter oligodendrocyte regeneration, we depleted cortical microglia by administration of chow supplemented with a CSF1R inhibitor following cuprizone-treatment. We found that more oligodendrocytes are formed during recovery from cuprizone treatment if reactive microglia are reduced. Collectively, these data suggest the reactive response of deep cortical microglia to demyelination serves as a barrier to remyelination at early stages of recovery from demyelination, and will importantly inform future strategies to target microglia to promote remyelination in MS.