As a first step towards a CAR-T-cell therapy targeting retinoblastoma, we here assessed CD171 expression in retrospective retinoblastoma samples and investigated the killing efficacy of CD171- and GD2-specific CAR-T cells harboring different spacer lengths (short versus long) and intracellular co-stimulatory domains (CD28 versus 4-1BB) in a panel of three different cell lines. Here, CD28 is linked to retinoblastoma.