Background Programmed cell death 1 (PD-1) receptor engagement on T cells by its ligand programmed cell death ligand 1 (PD-L1) is a key mechanism of immune escape, and antibody blockade of the interaction has emerged as an effective immunotherapeutic strategy in some cancers. The role and relevance of the PD-1 checkpoint in Ewing sarcoma (EwS) is not yet understood. Procedure Here, we investigated expression of PD-L1 and PD-1 in EwS by immunohistochemistry analysis of pretherapeutic tumor biopsies and in tumor xenografts following treatment with human T cells engineered to express a chimeric antigen receptor (CAR) against the tumor-associated antigen GD2. PD-L1 surface expression in EwS cell lines was assessed by flow cytometry. Results PD-L1 expression was not detectable on tumor cells in any of the 60 EwS biopsies. Infiltrating PD-L1 positive T cells were found in one tumor, and four biopsies contained PD-1-positive T cells. Of 13 EwS cell lines, none constitutively expressed PD-L1 on the cell surface. Interferon- cytokine stimulation induced upregulation of the ligand on all cell lines. Adoptive therapy with CAR gene-modified T cells in a mouse model did not induce PD-L1 expression in EwS xenografts despite tumor infiltration with PD-1+ CD3+ T cells. Conclusions EwS cells can upregulate PD-L1 under inflammatory conditions, but do not express the ligand in the pretherapeutic tumor microenvironment or postexposure to CAR T cells. PD-1 checkpoint blockade alone is thus unlikely to evoke potent immune responses against EwS. Identification of the relevant immune evasion strategies in EwS will be vital for the development of effective immune targeting strategies.