Severe retinal impairments, including blindness, affect millions worldwide, lowering the
quality of life and imposing a high economic burden. Most causative diseases with genetic backgrounds lack effective treatment, and research is being done to understand the pathology mechanisms and the risk factors. In vitro 3D cell culture systems (retinal organoids) gain popularity as a disease modeling platform because of their ability to reproduce retinogenesis, along with classical animal and 2D cell culture models. Additionally, there is a high need for new reporter pluripotent stem cell lines where cell populations critical for retinal development are visualized and accessible, such as retinal progenitor cells. When combined, retinal organoids derived from the reporter stem cell line might form an advantageous instrument for a new perspective into retinal development, disease modeling, and therapeutic studies. In the present work, we produce and characterize a new double reporter mouse embryonic stem cell line, Crx-GFP;Rax-mCherry. The Rax gene encodes an eye field transcription factor necessary for initiating and maintaining eye development. It is highly expressed in retinal progenitor cells and regulates their proliferation and cell type specification. Transcription factor CRX determines photoreceptor differentiation, maturation, and functioning. CRISPR/Cas9 double-nicking strategy was used to link reporter fluorescence gene mCherry to the last exon of the Rax gene. We proved the ability of the cell line to differentiate into retinal organoids and isolated the first Rax-mCherry-positive cells that appeared during retinal induction (Day 4 of differentiation). Transcriptomic, proteomic analysis, immunohistochemistry, and proliferation tests have confirmed the retinal progenitor commitment of isolated cells. Given this, the line allows the tracing and isolation of retinal progenitor (Rax+/Crx-) and photoreceptor cells (Crx+/Rax+ and Crx+/Rax-) from early to late stages of neural retina formation in retinal organoids and potentially, in stem-cell-derived mice. The line can find different applications in the context of developmental biology, disease modeling, and cell-based retinal regeneration therapies.