Canonical Wnt-β-catenin signalling is essential for skeletal muscle myogenesis during development, but its role in adult human skeletal muscle repair and regeneration remains unknown. Binding of Wnt ligands at the cell surface disables the capacity of GSK3 to phosphorylate β-catenin thus preventing its degradation and stimulating its interactions with TCF transcription factors. Here we manipulated a number of the molecular players in the Wnt signalling cascade in adult primary human skeletal muscle progenitors to assess their role in differentiation. Muscle biopsy samples were obtained from the vastus lateralis of healthy young male subjects (25.5±(SD) 3.1 years) following local anaesthesia (2% lidocaine). Muscle-derived cells were isolated and expanded in culture for 7 days after which CD56pos myogenic cells were immunomagnetically purified (Agley et al. 2013). Myogenic cells were either maintained in growth medium or stimulated to differentiate in serum-free conditions. In addition to studying β-catenin expression levels and localisation during differentiation, the following were applied in order to manipulate Wnt signalling: (i) pharmacological GSK inhibition, (ii) Lentiviral overexpression of constitutively active β-catenin (EβC) or (iii) dominant negative TCF4 (dnTCF4). Cells were analysed using western blotting, immunocytochemistry and qRT-PCR. Although detectable in growth medium, active β-catenin was mainly cytoplasmic. Under serum-free differentiating conditions active β-catenin stained strongly in the nucleus of differentiated MHCpos myotubes. Inhibition of GSK via BIO (5µM) increased active-β-catenin (Control: 45.8±8.0 (AU); BIO: 105.2±4.0 (AU); P<0.05), but severely blunted the normal differentiation response with marked and significant reductions in fusion index (Control: 73.1±3.7 %; BIO: 5.5±4.0%; P<0.001), individual myotube size and myogenin expression. Two further structurally diverse GSK inhibitors (CHIR-99021 and LiCl) produced very similar effects. EβC gave a milder phenotype of reduced fusion and myotube size although myogenin and MHC were still expressed. Contrastingly, loss of β-catenin-dependent TCF-driven transactivation (dnTCF4) entirely prevented fusion of myogenic precursors and myogenin expression was absent. Discrepancies between GSK3 inhibition and β-catenin overexpression reveal that active GSK3 is essential for myogenic fusion and differentiation with roles which likely extend beyond the regulation of β-catenin stability alone. Although greatly increasing nuclear β-catenin decreases myogenic cell fusion, disruption of its transcriptional co-activator role completely abolishes differentiation. Together these data show that active GSK3 and a functional β-catenin-TCF4 transcriptional complex are necessary for the differentiation of adult human myogenic progenitor cells.