During development, macrophages invade organs to establish phenotypically and transcriptionally distinct tissue-resident populations. How they invade and colonize these organs is unclear. In particular, it remains to be established whether they arise from naive equivalents that colonize organs randomly or whether there are committed macrophages that follow pre-determined migration paths. Here, by using a combination of genetics and imaging approaches in the zebrafish embryo, we have addressed how macrophages colonize the brain to become microglia. Identification and cloning of a mutant that lacks microglia has shown that Slc7a7, a Leucine/Arginine transporter, defines a restricted macrophage sub-lineage and is necessary for brain colonization. By taking a photoconversion approach, we show that these macrophages give rise to microglia. This study provides direct experimental evidence for the existence of sub-lineages among embryonic macrophages.