Microglia are the immune cells of the nervous system, where they act as resident macrophages during inflammatory events underlying many neuropathological conditions. Microglia derive from primitive myeloid precursors that colonize the nervous system during embryonic development. In the postnatal brain, microglia are initially mitotic, rounded in shape (amoeboid), and phagocytically active. As brain development proceeds, they gradually undergo a transition to a surveillant nonphagocytic state characterized by a highly branched (ramified) morphology. This ramification process is almost recapitulated in reverse during the process of microglia activation in the adult brain, when surveillant microglia undergo a ramified-to-amoeboid morphological transformation and become phagocytic in response to injury or disease. Little is known about the mechanisms controlling amoeboid microglial cell proliferation, activation, and ramification during brain development, despite the critical role of these processes in the establishment of the adult microglia pool and their relevance to microglia activation in the adult brain. Here we show that the mouse transcription factor Runx1, a key regulator of myeloid cell proliferation and differentiation, is expressed in forebrain amoeboid microglia during the first two postnatal weeks. Runx1 expression is then downregulated in ramified microglia. Runx1 inhibits mouse amoeboid microglia proliferation and promotes progression to the ramified state. We show further that Runx1 expression is upregulated in microglia following nerve injury in the adult mouse nervous system. These findings provide insight into the regulation of postnatal microglia activation and maturation to the ramified state and have implications for microglia biology in the developing and injured brain.