Oct4 and Nanog are two embryonic stem cell-specific transcription factors that play critical roles in the maintenance of ES cell pluripotency. In this study, we investigated the effects of Oct4 and Nanog expression on the differentiation state of myogenic cells which is sustained by a strong positive feedback loop. Oct4 and Nanog, either independently or simultaneously, were over-expressed in C2C12 myoblasts and the expression of myogenic lineage-specific genes and terminal differentiation were observed by RT-PCR. Over-expression of Oct4 in C2C12 cultures repressed, while exogenous Nanog did not significantly alter C2C12 terminal differentiation. The expression of Pax7 was reduced in all Oct4 over-expressing myoblasts and we identified a major Oct4 binding site in the Pax7 promoter. Simultaneous expression of Oct4 and Nanog in myoblasts inhibited the formation of myotubes, concomitant with a reduction in the endogenous levels of hallmark myogenic markers. Furthermore, over-expression of Oct4 and Nanog induced the expression of their endogenous counterparts along with the expression of Sox2. Using mammalian two-hybrid assays, we confirmed that Oct4 functions as a transcriptional repressor whereas Nanog functions as a transcriptional activator during muscle terminal differentiation. Importantly, in NOD SCID mice, the pluripotency of C2C12 cells was conferred by over-expression of Oct4 and Nanog. These results suggest that Oct4 in cooperation with Nanog strongly suppress the myogenic differentiation program and promotes pluripotency in myoblasts.