This study developed a modified monolayer culture technique for expansion of retinal progenitor cells in vitro. Using this method, spheres collected by low centrifugation from free floating cultures were re-seeded onto poly- D-lysine pre-coated flasks, and cultured in the retinal progenitor cells medium (including fibrous growth factor, epidermal growth factor and leukemia inhibitor factor) plus 5% FBS for the first 24 h, then FBS was removed. These cultured cells were proliferative, and in addition to expressing the neuroectodermal marker nestin they were multipotential. By immunocytochemistry and FACS analysis, we demonstrated that the percentage of nestin-expressing cells in passage 2 and passage 8 was 92.5 and 85.5%, respectively, which was greater than the percentage of their counterparts, 81.9 and 76.2%, in simple adhesive cultures (p < 0.05). Moreover, these cells preferentially differentiated into retinal neurons rather than glial cells.