Objective To establish a neuroglia progenitor differentiation system from human embryonic stem cells (hESCs) in a feeder layer- and serum-free medium. Methods hESCs grown in laminin coated culture plates were induced to form embryoid bodies (EBs) for 25 days. Then 25-day-old EBs were transferred to glial progenitor induction medium (GPIM) containing insulin, 5μg/L basic fibroblast growth factor (bFGF), 20μg/L epidermal growth factor (EGF) and 5μg/L triiodothyronine (T3) to continue culturing for 7-10 days. The differentiated fibroblast-like cells were digested and collected. The expressions of neuroglia progenitor genes were determined by RT-PCR. The special markers of neuroglia progenitors were examined by immunofluorescence staining. Results The differentiated cells were long spindle shaped. They expressed neuroglia progenitor special markers NG2, platelet\|derived growth factor receptor α（PDGFRα）. The percentage of NG2+, PDGFRα+ cells was 37.2% and 47.6%, respectively. They could further differentiate into astrocytes and oligodendroglias.Conclusion GPIM containing insulin, bFGF, EGF and T3 could induce hESCs differentiate into neuroglia progeni