Retinoblastoma is a childhood tumor of the retina that is caused mostly by biallelic inactivation of the tumor suppressor gene RB1. To generate a research resource, we abrogated expression of RB1 in H9 hESCs by CRISPR/Cas9 induced deletion of the RB1 promoter, either on one or on both alleles. This enables studies on the role of RB1 loss during differentiation, for example in differentiation towards neural retina. The generation of three isogenic lines per deletion state enables validation of phenotypic results in independent clonal lines.