Ewing sarcoma is primarily caused by a t(11;22) chromosomal translocation encoding the EWS-FLI1 fusion protein. To exert its oncogenic function, EWS-FLI1 acts as an aberrant transcription factor, broadly altering the gene expression profile of tumor cells. Nuclear factor-kappaB (NFkappaB) is a tightly regulated transcription factor controlling cell survival, proliferation and differentiation, as well as tumorigenesis. NFkappaB activity is very low in unstimulated Ewing sarcoma cells, but can be induced in response to tumor necrosis factor (TNF). We wondered whether NFkappaB activity could be modulated by EWS-FLI1 in Ewing sarcoma. Using a knockdown approach in Ewing sarcoma cells, we demonstrated that EWS-FLI1 has no influence on NFkappaB basal activity, but impairs TNF-induced NFkappaB-driven transcription, at least in part through inhibition of NFkappaB binding to DNA. We detected an in vivo physical interaction between the fusion protein and NFkappaB p65, which could mediate these effects. Our findings suggest that, besides directly controlling the activity of its primary target promoters, EWS-FLI1 can also indirectly influence gene expression in tumor cells by modulating the activity of key transcription factors such as NFkappaB.