Therapeutic application of pluripotent stem (PS) cell-derived products represents the ultimate goal of stem cell research. In order to apply this technology to patients, it is fundamental to characterize in detail the cell population of interest and identify strategies for its purification from unwanted cells using clinically-compatible methods. In the case of skeletal muscle wasting disorders, we have shown that human PS cell-derived PAX7-induced myogenic progenitors may represent an excellent candidate for cell therapy. To successfully translate this approach toward the clinic, we took advantage of next-generation sequencing techniques to dissect PAX7 function during human myogenesis. Combination of PAX7 genomic target profiling using ChIP-seq and whole transcriptome analysis (RNA-seq), in which we systematically evaluated different time points of the PAX7-dependent myogenic commitment from human PS cells, revealed a subset of genes differentially expressed at various stages of this differentiation process, including a discrete number of surface markers. After Fluorescence Activated Cell Sorting (FACS)-mediated screening, we identified α9β1 integrin, CD54 and Syndecan2 (SDC2), as potential surface markers to be used for the prospective isolation of human PS cell-derived myogenic progenitors. We demonstrate that these surface molecules reproducibly allow for the isolation of myogenic progenitors from multiple human ES/iPS cell lines, in both serum- and serum-free culture conditions, and that α9β1+CD54+SDC2+ (triple+) cells represent a homogenous population of PAX7+ cells endowed with in vivo muscle regeneration potential. Furthermore, we demonstrate that a single marker is sufficient for the magnetic-based isolation of myogenic progenitors, thus enabling adaptation of our differentiation protocol to cGMP standards. These novel findings provide a clinically relevant method for the purification of PS cell-derived muscle progenitors for clinical applications.