The proper formation and maintenance of myelin are essential for brain function. It has been found that myelination is regulated in a neuronal activity-dependent manner, resulting from interactions between neurons and oligodendrocytes. Recent reports also suggest that microglia play a crucial role in the formation and maintenance of myelin. However, the specifics of these microglia-myelin interactions remain largely unexplored. To investigate these interactions, we used a mouse cortical slice culture system. This system allowed us to visualize the tripartite relationship between microglia, neurons, and oligodendrocytes using tissue staining and live imaging techniques. This cortical slice culture system was set up by culturing slices from the cortex of 6-day-old mouse brains. Immunostaining of cultured sections with myelin and axon markers revealed that myelin forms on the axons, exhibiting a temporal progression comparable to that seen in mouse cortical sections in vivo. We then examined the impact of neuronal activity on microglia-myelin interactions by modulating neuronal activity using DREADD systems. Our results showed that changes in neuronal activity could have a bidirectional impact on the phagocytosis of myelin by microglia. Our live imaging further disclosed a myelin-like oligodendrocyte structure being phagocytosed by the microglia. In summary, our work has established a cortical slice culture system and suggested that neuronal activity influences the phagocytosis of myelin by microglia.