The identity of tumor-initiating cells in many cancer types is unknown. Tumors often express genes associated with embryonic development, although the contributions of zygotic programs to tumor initiation and formation are poorly understood. Here, we show that regeneration-induced loss of quiescence in p53-deficient muscle stem cells (MuSCs) results in rhabdomyosarcoma formation with 100% penetrance. Genomic analyses of purified tumor cells revealed spontaneous and discrete oncogenic amplifications in MuSCs that drive tumorigenesis, including, but not limited to, the amplification of the cleavage-stage Dux transcription factor (TF) Duxbl. We further found that Dux factors drive an early embryonic gene signature that defines a molecular subtype across a broad range of human cancers. Duxbl initiates tumorigenesis by enforcing a mesenchymal-to-epithelial transition, and targeted inactivation of Duxbl specifically in Duxbl-expressing tumor cells abolishes their expansion. These findings reveal how regeneration and genomic instability can interact to activate zygotic genes that drive tumor initiation and growth.