Although the majority of testicular germ cell tumors (TGCTs) are curable by cisplatin-based chemotherapy, in a few cases, the occurrence of cisplatin resistance results in a poor outcome. The biological basis of this differential cisplatin sensitivity in TGCTs remains largely unexplained. Embryonal carcinoma (EC) cells represent the presumptive tumor stem cells in nonseminomatous TGCTs and are known to express the embryonal transcription factor Oct-3/4 and to be hypersensitive to cisplatin. In the present study, we analyzed TGCT cell lines and nude mouse xenografts showing differential cisplatin sensitivity. Here we demonstrate that a lack of expression of Oct-3/4 in TGCT cells is associated with a higher apoptotic threshold and cisplatin resistance which is accompanied by an impaired caspase-9 activation, reduced caspase-3 activity and altered p53 accumulation. We were able to induce loss of Oct-3/4 in a cisplatin-sensitive EC cell line resulting in a secondary cisplatin-resistant cell type with retained EC cell characteristics and changes in apoptotic signaling identical to those in primary resistant cells. Furthermore, we show that EC cells are retained in their undifferentiated state by Oct-3/4 and that a complete and ultimate loss of Oct-3/4 followed by an early differentiation step is necessary to establish the cisplatin-resistant state. Our data suggest that loss of Oct-3/4 expression leads to induction of a higher apoptotic threshold and to cisplatin resistance in EC cells of nonseminomatous TGCTs. We hypothesize that in refractory TGCTs the original tumor stem cell population of Oct-3/4-positive, cisplatin-sensitive EC cells could be replaced by an Oct-3/4-negative, resistant population in a selection process. In contrast, the presence of the Oct-3/4-positive, highly sensitive EC cells as the tumor stem cell component in most TGCTs could explain the general high chemosensitivity and curability of these tumors.