Obtaining and manipulating neuronal cells are critical for neural biology basic mechanism studies and translational applications. Recent advances in protocol development and mechanism dissections have made direct induction of neuronal cells from other somatic cells (iN) a promising strategy for such purposes. In this study, we established a protocol to expand a population of fibroblast-like cells from adult human retinal tissues, which can be reprogrammed into iNs by forced expression of neurogenic transcription factors. Interestingly, the combination of Ascl1, Brn2, Myt1l, and NeuroD1 transcription factors, which has been demonstrated to be sufficient to reprogram human embryonic and dermal fibroblasts into iNs, failed to reprogram the fibroblast-like cells from human retinas into iNs. Instead, supplementing Ascl1 with Pax6 sufficed to convert the cells into iNs, which exhibited a typical neuronal morphology, expressed neural marker genes, displayed active and passive neuronal membrane activities, and made synaptic communications with other neurons. Moreover, iNs converted from retina-derived fibroblast-like cells contained high ratios of γ-Aminobutyric acid- (GABA-) and tyrosine hydroxylase- (TH-) positive neurons. Thus, the present study proposes a protocol that makes use of discarded retinal tissues from eye banks for iN generation, and suggests that different sources of somatic cells require different iN induction recipes and may also affect the iN subtype outputs. Our study may also facilitate the future development of methods to convert resident cells in situ into retinal neurons for treating retinal degeneration disease purpose.