Tissue-resident macrophages have important roles in tissue homeostasis and the immune response. The central nervous system is one of the first organs to become colonised with macrophages, and these then constitute the animal's microglia for its whole life. Because the factors underlying this early macrophage mobilisation are unclear, Philippe Herbomel and co-workers (p. 2797) performed a genetic screen to identify genes required for the establishment of microglia in zebrafish larvae. Their screen identified Trim33 (also known as transcriptional intermediary factor 1 gamma; Tif1-γ), a transcriptional regulator known to be involved in hematopoiesis and modulation of gene expression through transforming growth factor beta (TGF-β). The authors show that although primitive macrophages are produced normally in Trim33-deficient zebrafish embryos, their recruitment to the retina and the brain, the first epithelial tissues to become colonised by macrophages, is defective. Furthermore, as assessed here by 4D tracking of myeloid cells, Trim33-depleted macrophages and neutrophils display a reduced basal mobility in interstitial tissues and fail to respond to inflammatory signals. The role of Trim33 in macrophages appears to be conserved in mammals, as Trim33-deficient macrophages derived from mouse bone marrow also exhibit reduced 3D amoeboid motility in fibrous collagen gels. This work thus establishes Trim33 as a new factor involved in amoeboid motility of macrophages in vivo.