Retinoblastoma (RB) is a primary intraocular malignancy in childhood. Relapses may develop and cause secondary cancers during later development. This study was set up to identify optimal cell culture conditions for RB cell growth in vitro and to optimize tumor growth in an in vivo model. RB cell lines (Y79 and WERI-Rb1) were cultivated under three different in vitro conditions and apoptosis, proliferation and cell growth, as well as expression profiles of two epithelial-mesenchymal transition (EMT) markers, were analyzed. EMT gene expression profiles were not generally changed, whereas apoptosis levels, tumor cell proliferation, and in vitro growth were significantly influenced by different cell culture conditions. In order to optimize the time-limited chick chorioallantoic membrane (CAM) assay, we investigated two different time points of tumor cell inoculation (embryonic development day EDD8 and EDD10) as well as three different cell concentrations. We showed that inoculation at EDD8 led to decreased tumor formation and chicken viability, whereas different cell concentrations did not change size and weight of developing tumors. Our findings demonstrate that medium conditions in vitro as well as the starting point for CAM inoculation in ovo significantly influence the experimental outcome of investigations using RB cell lines.