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The importance of intratumoral heterogeneity has been highlighted by the identification and characterization of cancer stem cells (CSCs). Based on the differential responsiveness to a Sox2 reporter, SRR2, we had found a novel dichotomy in esophageal squamous cell carcinoma (ESCC) cells, with reporter‐responsive (RR) cells showing more CSC‐like features than reporter‐unresponsive (RU) cells. Specifically, RR cells exhibited significantly higher tumorsphere formation capacity, proportions of CD44 High  cells, chemoresistance to cisplatin, and tumorigenic potential in vivo. H 2 O 2 , a potent inducer of oxidative stress and reactive oxygen species, was found to induce a conversion from RU to RR cells; importantly, converted RR cells acquired CSC‐like features. The PI3K/AKT/c‐MYC signalling axis is important in this context, since pharmacologic blockade of PI3K‐AKT or siRNA knockdown of c‐MYC effectively inhibited the RR phenotype and its associated CSC‐like features, as well as the H 2 O 2 ‐induced RU/RR conversion. In a cohort of 188 ESCC patient samples, we found a significant correlation between strong c‐MYC expression and a short overall survival ( p  = .009). In conclusion, we have described a novel intratumoral heterogeneity in ESCC. The identification of the PI3K/AKT/c‐MYC axis as a driver of CSC‐like features carries therapeutic implications. S tem  C ells   2016;34:2040–2051

The PI3K/AKT/c‐MYC Axis Promotes the Acquisition of Cancer Stem‐Like Features in Esophageal Squamous Cell Carcinoma