Although the mechanisms that control growth are now well-understood, the mechanism that organisms use to assess their body size remains one of the great puzzles in biology. In insects undergoing complete metamorphosis, attainment of a threshold size specifies the final larval instar, after which growth stops and metamorphosis begins. We investigated the mechanism of threshold size assessment in the tobacco hornworm, Manduca sexta. The threshold size was found to vary with the amount of exposure to poor nutrient conditions whereas it was consistently lower under hypoxic conditions. Under these various conditions, the mass of the muscles plus integuments was correlated with the threshold size and myoglianin (myo) expression. Knockdown of myo in Tribolium castaneum led to larvae that stayed in the larval stage permanently even after passing the threshold size. We propose that increasing levels of Myo produced by the growing tissues allow larvae to assess their body size and trigger metamorphosis at the threshold size.