Once microglia arise from the progenitors in the yolk sac, these cells colonize the brain parenchyma in the embryonic stage. Border-associated macrophages (BAMs), the distinct cell type localized in the interface between the brain primordium and the vascular system, are derived from the same progenitors as microglia. Current studies have explored their lineage segregation outside the yolk sac, suggesting that BAMs seed microglia through their fate conversion during brain development. However, spatiotemporal evidence to support such microglial seeding and to explain how it occurs has not been provided. By the cell tracking analyses including intravital imaging, we found that intraventricular BAMs, which were abundantly observed along the inner surface of the cerebral wall, frequently enter the pallium at embryonic day (E) 12 in mice. Immunohistochemistry for the tracked cellsshowed that infiltrated BAMs in the pallium acquired microglial properties while losing a macrophage phenotype.We also found that the intraventricular BAMs are supplied from the roof plate.Our data suggest that “the roof plate-ventricle-pallium route” is an essential path for microglial colonization into the embryonic mouse brain. In this presentation, I will discuss the possible molecular mechanisms underlying microglial colonization.