Hyalocytes are macrophage-like cells residing in the eye vitreous cortex. Even though hyalocytes have been firstly described in the mid-Nineteenth century, they have been poorly explored. Recent researches highlighted hyalocyte involvement in both physiological and pathological processes of the vitreoretinal interface. Nonetheless, the majority of works involving hyalocyte cultures were carried out in animals, while fewer studies were performed on humans because their isolation requires vitrectomy. The aim of this study was to differentiate human induced pluripotent stem cells (iPSCs) into hyalocytes as a non-invasive method to continuously obtain cells. iPSCs were first differentiated into hematopoietic stem/progenitor cells (HSPCs) and then into macrophages. Macrophages were either left untreated (NT) or treated with ascorbic acid (AA) alone or combined with bFGF and/or TGF-β1. Additionally, macrophages were cultured in the presence of a pool of vitreous bodies from vitrectomies. Cells were analyzed for morphology and then for gene and protein expression through qRT-PCR, immunofluorescence, Western Blot, and flow cytometry. Similar to cells treated with the vitreous body, macrophages treated with AA alone or in combination with bFGF exhibited a more elongated shape compared to NT or cells treated with TGF-β1. Additionally, these treatments resulted in gene expression downregulation for S100A4, S100A10, S100B, and CX3CR1, while upregulating COL6A1, HLA-DRA, and CD74. At the protein level, S100B, CD14, and CD49d were downregulated with all treatments, while collagen VI and HLA-DR were upregulated. This work demonstrates that hyalocytes can be differentiated by treatment of iPSC-derived macrophages with ascorbic acid for a period of 21 days.