The introduction of stem cell-based technologies for the derivation of 
three-dimensional retinal tissues, the so-called retinal organoids, offers many 
new possibilities for vision research: Organoids facilitate studies on retinal 
development and in vitro retinal disease modeling, as well as being valuable for 
drug testing. Further, retinal organoids also provide an unlimited cell source 
for cell replacement therapies. Here, we describe our protocol for efficiently 
differentiating large, stratified retinal organoids from mouse embryonic stem 
cells: unbiased manual dissection of the developing retinal organoid at an early 
stage into three evenly sized neuroepithelial portions (trisection step) doubles 
the yield of high-quality organoids. We also describe some useful applications 
of the protocol, e.g., generation of rod- or cone-enriched retinal organoids, 
AAV transfection, and cell birth dating. In addition, we provide details of how 
to process retinal organoids for single organoid gene expression analysis, 
immunohistochemistry, and electron microscopy.