Ewing sarcoma is a bone and soft-tissue tumor that depends on the activity of the EWS-FLI1 transcription factor for cell survival. Although a number of compounds have been shown to inhibit EWS-FLI1 in vitro, a clinical EWS-FLI1-directed therapy has not been achieved. One problem plaguing drug development efforts is the lack of a suitable, non-invasive, pharmacodynamic marker of EWS-FLI1 activity. Here we show that F-FLT PET (F- 3'-deoxy-3'-fluorothymidine positron emission tomography) reflects EWS-FLI1 activity in Ewing sarcoma cells both in vitro and in vivo. F-FLT is transported into the cell by ENT1 and ENT2, where it is phosphorylated by TK1 and trapped intracellularly. In this report, we show that silencing of EWS-FLI1 with either siRNA or small-molecule EWS-FLI1 inhibitors suppressed the expression of ENT1, ENT2, and TK1 and thus decreased F-FLT PET activity. This effect was not through a generalized loss in viability or metabolic suppression, as there was no suppression of F-FDG PET activity and no suppression with chemotherapy. These results provide the basis for the clinical translation of F-FLT as a companion biomarker of EWS-FLI1 activity and a novel diagnostic imaging approach for Ewing sarcoma.