Microglia are known to play essential roles in the development, progression and treatment of diverse neurodegenerative diseases in the central nervous system, including the retina, brain and spinal cord. Recently, brain-induced microglia-like cells (iMGs) have been generated from human pluripotent stem cells (hPSCs); however, retinal microglia have yet to be developed in vitro. In this study, by mimicking in vivo microglial development, we established a simplified approach to differentiate hPSCs into high purity (>90%) iMGs. The iMGs express microglia-specific markers, release cytokines upon stimulation, and are capable of phagocytizing bacteria. When co-cultured with three-dimensional human retinal organoids (hROs), iMGs migrated into the hROs, tended to differentiate into resident retinal microglia, and simultaneously induced apoptosis in some neural cells. Notably, the resident iMGs in the hROs formed sparse web-like structures beneath the photoreceptor cell layer, resembling microglia's orientation in human retina. In conclusion, we developed a simplified and efficient method to generate microglia from human pluripotent stem cells, and we report the first derivation of retinaresident microglia in vitro, providing a new source of human retinal microglia for developmental and disease studies and regenerative therapeutics.