Callosal projection neurons, one of the major types of projection neurons in the mammalian cerebral cortex, require neuronal activity for their axonal projections [H. Mizuno et al. (2007) J. Neurosci., 27, 6760-6770; C. L. Wang et al. (2007) J. Neurosci., 27, 11334-11342]. Here we established a method to label a few callosal axons with enhanced green fluorescent protein in the mouse cerebral cortex and examined the effect of pre-synaptic/post-synaptic neuron silencing on the morphology of individual callosal axons. Pre-synaptic/post-synaptic neurons were electrically silenced by Kir2.1 potassium channel overexpression. Single axon tracing showed that, after reaching the cortical innervation area, green fluorescent protein-labeled callosal axons underwent successive developmental stages: axon growth, branching, layer-specific targeting and arbor formation between post-natal day (P)5 and P9, and the subsequent elaboration of axon arbors between P9 and P15. Reducing pre-synaptic neuronal activity disturbed axon growth and branching before P9, as well as arbor elaboration afterwards. In contrast, silencing post-synaptic neurons disturbed axon arbor elaboration between P9 and P15. Thus, pre-synaptic neuron silencing affected significantly earlier stages of callosal projection neuron axon development than post-synaptic neuron silencing. Silencing both pre-synaptic and post-synaptic neurons impaired callosal axon projections, suggesting that certain levels of firing activity in pre-synaptic and post-synaptic neurons are required for callosal axon development. Our findings provide in-vivo evidence that pre-synaptic and post-synaptic neuronal activities play critical, and presumably differential, roles in axon growth, branching, arbor formation and elaboration during cortical axon development.