Background: Chimeric antigen receptor (CAR) T cell therapy of pediatric sarcomas is challenged by the paucity of targetable cell surface antigens. A candidate target in osteosarcoma (OS) is the ganglioside GD2, but heterogeneous expression limits its value. We aimed to identify mechanisms that upregulate GD2 expression in OS. Methods: Surface expression of GD2 in OS cell lines was assessed by flow cytometry. Transcription and translation of genes encoding for enzymes involved in GD2 synthesis were studied by quantitative real-time PCR and by using a construct containing the 5’ untranslated region (5’UTR) of the GD3 synthase-mRNA. An in vitro cytotoxicity assay was used to study the sensitivity of OS cells to targeting by GD2-specific CAR T cells. Results: GD2 surface expression varies both among and within individual OS cell lines. Pharmacological approaches, including inhibition of the histone methyltransferase Enhancer of Zeste Homolog 2 (EZH2) and modulation of the protein kinase C (PKC), failed to increase GD2 expression. Instead, cell confluency was associated with higher GD2 expression levels both in monolayer cultures and in tumor spheroids and with increased sensitivity to in vitro cytolysis by GD2-specific CAR T cells. Confluency-dependent upregulation of GD2 expression in OS cells is mediated by increased de novo biosynthesis through a yet unknown mechanism. Conclusions: Expression of GD2 in OS is highly variable and associated with increasing cell confluency in vitro. Strategies for selective upregulation of GD2 are needed to enable effective therapeutic targeting of this antigen in OS.