The core embryonic stem cell transcription factors (TFs) OCT3/4 (OCT4), NANOG, and SOX2 have shared as well as nonoverlapping roles in stem cell growth and differentiation. These same TFs are also expressed in various types of human germ cell tumors (GCTs), implicating them in regulation of tumor growth and differentiation. Although NANOG and OCT3/4 are sensitive and specific markers for seminoma and embryonal carcinoma, neither factor aids in the clinically important distinction of seminomatous from nonseminomatous tumors. In contrast, expression profiling data suggest that SOX2 may help with this distinction. To determine if a panel of embryonic stem cell TFs (NANOG, OCT3/4, and SOX2) can facilitate the identification and distinction of seminomatous from nonseminomatous GCTs, we evaluated their expression by immunohistochemistry in primary testicular (n=41) and metastatic retroperitoneal (n=43) GCTs. Our results confirm NANOG and OCT3/4 as sensitive and specific markers for primary seminoma and embryonal carcinoma and demonstrate the novel finding that NANOG is a marker for metastatic GCTs. In addition, SOX2 is expressed in embryonal carcinoma but not pure seminoma and is therefore a useful diagnostic marker for distinguishing seminomatous and nonseminomatous GCTs. In summary, we find that the embryonic stem cell TF signature of seminoma is NANOG+, OCT3/4+, and SOX2−, whereas embryonal carcinoma is NANOG+, OCT3/4+, and SOX2+, and expect these immunohistochemical profiles will facilitate the diagnosis of both primary and metastatic GCTs.