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Provided is a culture medium and a method of differentiating a pluripotent stem cell into a microglial cell by using the culture medium, wherein the culture medium comprises amino acids, vitamins, inorganic salts, glucose or galactose, buffering agent, serum albumin, transferrin, sodium chloride, a pyruvate salt, glutamine, biotin, ascorbic acid, and lactic acid, and the osmolality of the culture medium is at least 275 mOsm. The culture medium may further comprise corticosterone, linoleic acid, linolenic acid, progesterone, tocopherol, lipoic acid, L-carnitine, ethanolamine, D-galactose, putrescine dihydrochloride, sodium selenite, BSA, catalase, glutathione, insulin, human-holo-transferrin, superoxide dismutase, and T3.