The encouraging response and improved survival of acute promyelocytic leukemia patients following retinoic acid treatment has rendered differentiation therapy an attractive option in cancer treatment. Given that terminal differentiation represents a considerable barrier in retinoblastoma tumorigenesis and that retinoblastoma has a significantly higher spontaneous degeneration rate compared with other tumors (1,000-fold change), differentiation therapy represents a promising alternative in the treatment of retinoblastoma. However, the full differentiation potential of retinoblastoma still unknown. The present study was designed to investigate the extend differentiation of the classical retinoblastoma cell line WERI-Rb-1 (W-RBCs). Several critical cell signaling pathways and key genes related to cell proliferation and differentiation were comprehensively regulated to control the fate of W-RBCs. Various strategies were applied to optimize simple and time-saving methods to induce W-RBCs into different types of retinal neuron-like cells (RNLCs) in vitro. Further, the tumorigenesis of these differentiated W-RBCs was tested in nude mice in vivo. W-RBCs were found to inherently express both retinal progenitor cell-and embryonic stem cell-related genes or proteins. Moreover, the addition of antagonists of critical cell signals (Wnt, Nodal, BMP4 and Notch), even without atonal bHLH transcription factor 7 gene transfection, could directly induce W-RBCs into RNLCs, and especially into photoreceptor-like and retinal ganglion-like cells. Interestingly, the differentiated cells showed remarkably poorer tumorigenesis in vivo. These findings may offer new insights on the oriented differentiation of W-RBCs into RNLCs with low tumorigenicity and provide potential targets for retinoblastoma differentiation therapy.