Recent advances in self-organizing, 3-dimensional tissue cultures of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) provided an invitro model that recapitulates many aspects of the invivo developmental steps. Using Rax-GFP-expressing ESCs, newly generated Six3-/- iPSCs, andconditional null Six3delta/f;Rax-Cre ESCs, we identified Six3 repression of R-spondin 2 (Rspo2) as a required step during optic vesicle morphogenesis and neuroretina differentiation. We validated these results invivo by showing that transient ectopic expression of Rspo2 in the anterior neural plate of transgenic mouse embryos was sufficient to inhibit neuroretina differentiation. Additionally, using a chimeric eye organoid assay, we determined that Six3 null cells exert a non-cell-autonomous repressive effect during optic vesicle formation and neuroretina differentiation. Our results further validate the organoid culture system as a reliable and fast alternative to identify and evaluate genes involved in eye morphogenesis and neuroretina differentiation invivo.