How myoblasts differentiate and fuse to form myotubes has important implications for skeletal muscle development, regeneration and hypertrophy. The majority of muscle cell lines, in vitro, are grown to confluence in high serum media and are differentiated by reducing the amount of growth factors present by serum withdrawal. The combination of high cell density and low serum are necessary to induce differentiation and fusion. However, despite the wide spread use of the procedure, the mechanisms by which this induces differentiation are not fully understood. One would expect that serum withdrawal would decrease growth factor receptor mediated signalling. However, during the time course of differentiation the opposite occurs. PKB activity as measured by GSK3α/β phosphorylation increases by 2.5±1.5, 3.1±0.3 and 3.38±1.3 fold at day 1, 3 and 5 respectively. The increase in PKB activity at day 1 occurs concomitant with a 2.2±0.4 fold increase in IRS-1 expression and a 2.4±0.5 fold decrease in S6K-1 activity, as measured by S6 phosphorylation. Considering the role of S6K1 in IRS-1 regulation this decrease in S6K1 activity may initiate the fusion process. To resolve the role of S6K1 in differentiation we utilized two separate expression systems; one under the control of the myosin light chain 2-fast (MLC2-F) promoter, and the other under the control of the RheoSwitch inducer to overexpress various mutants of S6K1. When kinase dead S6K1 is overexpressed just before differentiation using the MLC2-F promoter, myoblast fusion is enhanced, as is myosin heavy chain (MHC) expression (a marker of terminal differentiation). However when wild type S6K1 is overexpressed with the same system, fusion and myosin heavy chain expression are inhibited. When the expression of these genes is induced at the same time as differentiation, fusion appears to be normal. However, myosin heavy chain expression is enhanced in the cells overexpressing the kinase dead S6K1 and inhibited in the cells overexpressing wild type S6K1 suggesting that high S6K1 activity inhibits differentiation. The more widely recognised role of S6K1 in cell size regulation is also observed in these cells. The cells overexpressing kinase dead S6K1 did not achieve the same diameter as transfection controls in spite of greater differentiation and MHC expression. In conclusion, the dynamic changes in S6K1 activity during differentiation are required for normal myoblast differentiation likely through its regulation of IRS-1 levels. Post fusion, the increase of S6K1 activity is required for the attainment of normal cell size.