Although molecular testing can definitively distinguish Ewing sarcoma (EWS) from synovial sarcoma (SS) it is frequently desirable to provide a confident preliminary diagnosis before such analysis can be completed. Recently, the nuclear markers NKX2.2 and TLE1 have been shown to have good sensitivity but imperfect specificity, respectively, for EWS and SS. However, the performance of these markers has not been extensively evaluated within this specific differential diagnosis. This study performed NKX2.2, TLE1, and CD99 immunohistochemistry in a group of EWS and SSs confirmed by reverse transcription-polymerase chain reaction to evaluate the utility of these novel markers in this context. NKX2.2 staining was overall 75% sensitive and 91.7% specific for EWS and was never seen in SS. Although the specificity of TLE1 staining was impacted by antibody used, it was at best only 75% specific for SS. However, a lack of reactivity had a 100% negative predictive value against a SS diagnosis. Overall, immunohistochemistry for NKX2.2 and TLE1 can provide a useful first step in helping to distinguish EWS and SS.