<p>Rhabdomyosarcoma (RMS) is an aggressive cancer thought to arise from impaired myogenesis. This can be substantially overcome by increasing the levels of pannexin 1 (PANX1), a critical component of the myogenic program, but the mechanism involved is unknown. Using RNA-seq, we have previously found that overexpression of PANX1 dramatically reshapes the transcriptomic landscape of RMS including downregulation of a myogenic modulator, <i>APOBEC2</i> (apolipoprotein B mRNA editing enzyme catalytic subunit 2). Following this clue, we investigated the role of APOBEC2 in the PANX1-mediated suppression of RMS malignancy. Here we show that, using a panel of patient-derived RMS cell lines and tumor specimens, APOBEC2 is expressed in RMS, but that its levels are lower than those in both differentiating myoblasts and skeletal muscle. In most RMS cell lines examined, APOBEC2 accumulates during proliferation and sustains their stem-like characteristics, as evidenced by its ability to promote the growth of spheroids upon increased expression. Yet, ectopic PANX1 expression led to a marked downregulation of APOBEC2 across a large proportion of RMS cell lines assessed. Strikingly, these were the same cells in which PANX1 triggers multinucleation. We further reveal that, like healthy myoblasts progressing through myogenesis, the multinucleation observed here in RMS cells results from cell fusion. Importantly, in RMS cells engineered to overexpress APOBEC2, PANX1 no longer enhances cell fusion, but its other anti-tumorigenic properties are still preserved. Collectively, our data indicate that PANX1 promotes RMS cell fusion by downregulating APOBEC2 expression, driving these tumor cells further into the myogenic program.</p>