Recently, we have demonstrated that F3/contactin and NB-3 are trans-acting extracellular ligands of Notch that promote differentiation of neural stem cells and oligodendrocyte precursor cells into mature oligodendrocytes (OLs). Here, we demonstrate that human bone marrow stromal cells (hBMSCs) can be induced to differentiate into cells with myelinating glial cell characteristics in mouse retina after predifferentiation in vitro. Isolated CD90(+) hBMSCs treated with beta-mercaptoethanol for 1 day and retinoic acid for 3 days in culture changed into myelinating glia-like cells (MGLCs). More cells expressed NG2, an early OL marker, after treatment, but expression of O4, a mature OL marker, was negligible. Subsequently, the population of O4(+) cells was significantly increased after the MGLCs were predifferentiated in culture in the presence of either F3/contactin or multiple factors, including forskolin, basic fibroblast growth factor, platelet-derived growth factor, and heregulin, in vitro for another 3 days. Notably, 2 months after transplantation into mouse retina, the predifferentiated cells changed morphologically into cells resembling mature MGLCs and expressing O4 and myelin basic protein, two mature myelinating glial cell markers. The cells sent out processes to contact and wrap axons, an event that normally occurs during early stages of myelination, in the retina. The results suggest that CD90(+) hBMSCs are capable of morphological and functional differentiation into MGLCs in vivo through predifferentiation by triggering F3/Notch signaling in vitro.