Living cells assemble their actin networks by regulating reactions at the barbed end of actin filaments. Formins accelerate elongation, capping protein (CP) arrests growth and twinfilin promotes depolymerization at barbed ends. How cells integrate these disparate activities within a shared cytoplasm to produce diverse actin networks, each with distinct morphologies and finely tuned assembly kinetics, is unclear. We used microfluidics-assisted TIRF microscopy to investigate how formin mDia1, CP and twinfilin influence the elongation of actin filament barbed ends. We discovered that the three proteins can simultaneously bind a barbed end in a multiprotein complex. Three-color single molecule experiments showed that twinfilin cannot bind actin filament ends occupied by formin mDia1 unless CP is present. The trimeric complex is short-lived (∼1s) and results in rapid dissociation of CP by twinfilin causing resumption of rapid formin- based elongation. Thus, the depolymerase twinfilin acts as a pro-formin factor that promotes polymerization when both CP and formin are present. While a single twinfilin binding event is sufficient to displace CP from the trimeric complex, it takes about 30 independent twinfilin binding events to remove capping protein from CP-bound barbed end. Our findings establish a new paradigm in which polymerases, depolymerases and cappers work in concert to tune cellular actin assembly.