Retinal neurons and glia in the adult vertebrate retina are differentiated from multipotent retinal progenitors in the eyecups under the regulation of intrinsic and extrinsic factors, but the molecular mechanism underlying the process is partially understood. Functional studies using engineered mice provide tremendous insight into the mechanisms of retinal cell differentiation, but in utero embryogenesis prevents manipulations of mouse embryonic retina. Mouse eyecup culture using a culture filter or insert has been developed, but retinal structure is often altered due to the flattening of mouse eyecups in these culture systems. In this chapter, we describe three-dimensional culture of embryonic mouse eyecups. In our system, cell differentiation, stratified retinal structure, and ciliary margins in cultured eyecups were reminiscent of those in vivo. Our 3D culture of mouse eyecups has multiple applications when wild-type or engineered mice are used as models for studying retinal cell differentiation.