The embryonic stem cell transcription factors SOX2, NANOG, and OCT3/4 are involved in the regulation of germ cell tumor growth and differentiation. They, and D2-40 (podoplanin), an antigen expressed in seminomas, are emerging as useful diagnostic markers in testicular germ cell tumors. This study evaluates the use of these markers in ovarian tumors. Ovarian germ cell tumors (n=31) have distinct immunostaining profiles, depending on the type of differentiation as follows: dysgerminoma (SOX2-, NANOG+, OCT3/4+, D2-40+), embryonal carcinoma (SOX2+, NANOG+, OCT3/4+, D2-40-), immature teratomas (SOX2+, NANOG-, OCT3/4-, D2-40-), yolk sac tumors, and choriocarcinoma (SOX2-, NANOG-, OCT3/4-, D2-40-). In immature teratomas, SOX2 positivity was limited to neural and epithelial tissues, and OCT3/4 was positive only in scattered epithelial cells (<10% of cells). Nongerm cell tumors (n=57, including surface-epithelial stromal tumors and sex-cord stromal tumors) were negative for NANOG and D2-40. OCT3/4 was positive in 4 of 9 adult granulosa cell tumors (15% to 85% of cells). In a small number of surface-epithelial stromal tumors, SOX2 and/or OCT3/4 were variably positive (20% to 90% of cells). Of the markers, SOX2 and D2-40 discriminated between dysgerminoma and embryonal carcinoma. NANOG distinguished between either of these 2 tumors and nongerm cell tumors. The inclusion of these markers should therefore be considered in cases of pure or mixed ovarian germ cell tumors that are difficult to classify, and to exclude nongerm cell tumor mimics.