Verification that cell lines used for cancer research are derived from malignant cells in primary tumors is imperative to avoid invalidation of study results. Retinoblastoma is a childhood ocular tumor that develops from loss of functional retinoblastoma protein (pRb) as a result of genetic or epigenetic changes that affect both alleles of the RB1 gene. These patients contain unique identifiable genetic signatures specifically present in malignant cells. Primary cultures derived from retinoblastoma tumors can be established as non-adherent tumorspheres when grown in defined media or as attached monolayers when grown in serum-containing media. While the RB1 genotypes of tumorspheres match those of the primary tumor, adherent cultures have the germline RB1 genotype. Tumorspheres derived from pRb-negative tumors do not express pRb and express the neuroendocrine tumor markers synaptophysin and microtubule-associated protein 2 (MAP2). Adherent cells are synaptophysin-negative and express pRb, the epithelial cell marker cytokeratin that is expressed in the retinal pigmented epithelium and the vascular endothelial cell marker CD34. While tumorspheres are of malignant origin, our results cast doubt on the assumption that adherent tumor-derived cultures are always valid in vitro models of malignant cells and emphasize the need for validation of primary tumor cultures.