Hepatoblastoma (HB) is the most common liver malignancy observed in childhood. In spite of the clinical advancements, the poor prognosis in high-risk patients dictates the development of preclinical models that precisely reflect the disease. This study seeks to evaluate the tumorigenic capacity and reliability of the extensively used HepG2 cell line in a bioluminescence imaging-supported orthotopic mouse model. Lentivirally engineered HepG2-Luc cells were injected into the livers of athymic nude mice which were grouped based on cell dosage (2.5–3.0 million) with or without Matrigel (+/-). Tumor development was monitored for 6 weeks using the IVIS system, and histopathological confirmation was performed at the end of the experiment. In vitro analyses confirmed that HepG2-Luc cells produced approximately 3500-fold stronger signals than the parental cells. However, overall tumor engraftment remained limited in vivo. Histopathological examination identified tumor foci in 20% of the 3 million (+) group and 16.7% of the 2.5 million (−) group. Matrigel supplementation did not significantly affect engraftment rates. In some animals, detectable radiometric signals were recorded despite the absence of a tissue mass, which was attributed to the dispersion of cells as microscopic foci and/or peritoneal leakage. The results demonstrate, for the first time in the literature, that HepG2 cells have low tumorigenic potential in J:NU mice. For liver cancer research, using more aggressive cell lines such as Huh-6, Huh-7, Hep-3B or employing more profoundly immunodeficient mouse strains, may represent a more efficient approach.