Axonal branching is thought to be regulated not only by genetically defined programs but also by neural activity in the developing nervous system. Here we investigated the role of pre- and postsynaptic activity in axon branching in the thalamocortical (TC) projection using organotypic coculture preparations of the thalamus and cortex. Individual TC axons were labeled with enhanced yellow fluorescent protein by transfection into thalamic neurons. To manipulate firing activity, a vector encoding an inward rectifying potassium channel (Kir2.1) was introduced into either thalamic or cortical cells. Firing activity was monitored with multielectrode dishes during culturing. We found that axon branching was markedly suppressed in Kir2.1-overexpressing thalamic cells, in which neural activity was silenced. Similar suppression of TC axon branching was also found when cortical cell activity was reduced by expressing Kir2.1. These results indicate that both pre- and postsynaptic activity is required for TC axon branching during development.