MicroRNAs have a critical role in oligodendrocyte development including cell proliferation, differentiation, and myelin formation. MicroRNA 338 (miR-338) is necessary to promote oligodendrocyte differentiation by repressing negative regulators of oligodendrocyte differentiation. Oligodendrocytes (OLs) are responsible for myelin sheath synthesis around nerve fibers in the central nervous system (CNS) that form the myelin sheath of axons. Human endometrial-derived stromal cells (hEnSCs) are a new source of mesenchymal-like stem cells for cell replacement therapy. The hEnSCs, after treating with fibroblast growth factor 2/epidermal growth factor (20 ng/mL) and platelet-derived growth factor (PDGF)-AA (10 ng/mL) for 12 days, were divided in two groups: in the first group, the cells were treated by triiodothyronine (T3), and in the second group, the cells were infected by miR-338-green fluorescent protein-expressing lentiviruses. Six days after infection, the cells were collected and analyzed for the expression of stage-specific markers Nestin, microtubule-associated protein 2, neurofilament-L, oligodendrocyte lineage transcription factor, SRY-box containing gene 10, PDGF receptor alpha, 2',3'-cyclic nucleotide 3' phosphodiesterase, A2B5, O4, and myelin basic protein by immunocytochemistry and quantitative reverse transcription PCR. Result showed that in the infected cells, the expression of pre-oligodendrocyte markers was higher than that of T3-treated cells. The EnSCs can differentiate to oligodendrocyte cells by the overexpression of miR-338, and these cells can be used as a unique source for cell therapy of neurodegenerative disease.