Myostatin is a negative regulator of skeletal muscle size, previously shown to inhibit muscle cell differentiation. Myostatin requires both Smad2 and Smad3 downstream of the activin receptor II (ActRII)/activin receptor-like kinase (ALK) receptor complex. Other transforming growth factor-beta (TGF-beta)-like molecules can also block differentiation, including TGF-beta(1), growth differentiation factor 11 (GDF-11), activins, bone morphogenetic protein 2 (BMP-2) and BMP-7. Myostatin inhibits activation of the Akt/mammalian target of rapamycin (mTOR)/p70S6 protein synthesis pathway, which mediates both differentiation in myoblasts and hypertrophy in myotubes. Blockade of the Akt/mTOR pathway, using small interfering RNA to regulatory-associated protein of mTOR (RAPTOR), a component of TOR signaling complex 1 (TORC1), increases myostatin-induced phosphorylation of Smad2, establishing a myostatin signaling-amplification role for blockade of Akt. Blockade of RAPTOR also facilitates myostatin's inhibition of muscle differentiation. Inhibition of TORC2, via rapamycin-insensitive companion of mTOR (RICTOR), is sufficient to inhibit differentiation on its own. Furthermore, myostatin decreases the diameter of postdifferentiated myotubes. However, rather than causing upregulation of the E3 ubiquitin ligases muscle RING-finger 1 (MuRF1) and muscle atrophy F-box (MAFbx), previously shown to mediate skeletal muscle atrophy, myostatin decreases expression of these atrophy markers in differentiated myotubes, as well as other genes normally upregulated during differentiation. These findings demonstrate that myostatin signaling acts by blocking genes induced during differentiation, even in a myotube, as opposed to activating the distinct "atrophy program." In vivo, inhibition of myostatin increases muscle creatine kinase activity, coincident with an increase in muscle size, demonstrating that this in vitro differentiation measure is also upregulated in vivo.