The diagnosis of testicular germ cell tumors (GCTs) sometimes can be challenging without ancillary markers. Here we performed an immunohistochemical study of a novel stem cell marker SALL4 in a large series of 110 primary testicular GCTs (65 pure and 45 mixed) containing the following types of tumors and/or tumor components: 50 intratubular germ cell neoplasias (ITGCNs), 62 classic seminomas, 2 spermatocytic seminomas, 39 embryonal carcinomas (EC), 5 pediatric and 26 postpubertal yolk sac tumors (YST), 7 pediatric and 25 postpubertal teratomas, and 5 choriocarcinomas. We compared SALL4 with OCT4 in all GCTs, and SALL4 to alpha-fetoprotein (AFP) and glypican-3 in all YSTs. To test SALL4 specificity, 23 testicular non-GCTs (10 Leydig cell tumors, 4 Sertoli cell tumors, 3 adenomatoid tumors, 3 paratesticular rhabdomyosarcomas, 2 diffuse large B-cell lymphomas, and 1 rete testis papillary cystadenoma) and 275 nontesticular tumors (158 metastatic carcinomas, 12 metastatic melanomas, 11 primary and 2 metastatic mesotheliomas, and 72 primary and 20 metastatic sarcomas) were also stained for SALL4. All ITGCNs, classic seminomas, and ECs demonstrated strong SALL4 and OCT4 staining in more than 90% tumor cells. All 31 YSTs (5 pediatric and 26 postpubertal) showed strong positive SALL4 staining in more than 90% tumor cells but had negative OCT4 staining. Both spermatocytic seminomas showed positive SALL4 staining in 80% to 95% tumor cells in all 3 types of tumor cells with weak-to-moderate staining intensity. Mononucleated trophoblastic cells were variably positive for SALL4 staining in all 5 choriocarcinomas. Focal SALL4 staining was seen in 4 of 7 pediatric and 23 of 27 postpubertal teratomas. OCT4 staining was not seen in any spermatocytic seminoma, choriocarcinoma, or teratoma. No SALL4 staining was seen in all 23 testicular non-GCTs. Of 275 nontesticular tumors, only 10 carcinomas and 1 sarcoma showed focal (<25% tumor cells) weak SALL4 staining. The only non-neoplastic cells within the testis stained with SALL4 were spermatogonia and few primary spermatocytes. AFP staining was seen in 29 of 31 YST but it was often focal and patchy. Although all 31 YSTs showed glypican-3 staining, 14 (45%) show staining in less than 30% tumor cells. Our findings indicate that SALL4 is a novel sensitive and relatively specific marker for testicular GCTs. SALL4 is a more sensitive marker than AFP and glypican-3 for YST.