Cell migration is a rate-limiting event in skin wound healing. In unwounded skin, cells are nourished by plasma. When skin is wounded, resident cells encounter serum for the first time. As the wound heals, the cells experience a transition of serum back to plasma. In this study, we report that human serum selectively promotes epidermal cell migration and halts dermal cell migration. In contrast, human plasma promotes dermal but not epidermal cell migration. The on-and-off switch is operated by transforming growth factor (TGF) β3 levels, which are undetectable in plasma and high in serum, and by TGFβ receptor (TβR) type II levels, which are low in epidermal cells and high in dermal cells. Depletion of TGFβ3 from serum converts serum to a plasmalike reagent. The addition of TGFβ3 to plasma converts it to a serumlike reagent. Down-regulation of TβRII in dermal cells or up-regulation of TβRII in epidermal cells reverses their migratory responses to serum and plasma, respectively. Therefore, the naturally occurring plasma→serum→plasma transition during wound healing orchestrates the orderly migration of dermal and epidermal cells.