Previous studies have demonstrated that a small population of cancer stem cell-like cells exists in retinoblastoma. To provide a model for studying this population, we sought to establish a long-term culture from human retinoblastoma that have cancer stem cell-like properties. Fresh tumor tissue was digested and cultured in serum-free medium. Tumor spheres formed and were passaged continuously. Stem cell properties were examined through immunostaining, real-time quantitative RT-PCR and chemoresistance assay. Tumorigenicity of the tumor sphere-forming cells was confirmed by xenograft experiments. Furthermore, we examined the expression of cell surface markers CD44 and CD133. Tumor cells expanded as floating spheres for more than 30 passages. Sphere-forming cells overexpressed stem cell genes Oct‑4, Nestin and Pax6. Immunostaining of spheres showed positivity for Nestin, Pax6 and also ABCG2. In contrast, differentiated cells derived from these spheres expressed high levels of mature retinal cell markers MAP2, GFAP, recoverin, Opsin B and Nrl, and showed immunoreactivity for NF200, GFAP, recoverin and PKCα. Furthermore, both CD44 and CD133 were highly expressed in sphere-forming cells vs. differentiated cells. Sphere-forming cells displayed higher chemoresistance to carboplatin as opposed to differentiated cells. Moreover, intraocular injection of as few as 2x103 sphere-forming cells into NOD/SCID mice gave rise to new tumors similar to the original patient tumors. These results revealed that the sphere-forming cells preserved their stem cell properties and tumorigenicity, even after long-term culture. This would be a suitable in vitro model to study cancer stem-like cells in retinoblastoma and to develop chemotherapeutic drugs and strategies.