In many CNS pathways, target innervation occurs by axon branching rather than extension of the primary growth cone into targets. To investigate mechanisms of branch formation, we studied the effects of attractive and inhibitory guidance cues on cortical axon branching. We found that netrin-1, which attracts cortical axons, and FGF-2 increased branching by >50%, whereas semaphorin 3A (Sema3A), which repels cortical axons, inhibited branching by 50%. Importantly, none of the factors affected axon length significantly. The increase in branching by FGF-2 and the inhibition of branching by Sema3A were mediated by opposing effects on the growth cone (expansion vs collapse) and on the cytoskeleton. FGF-2 increased actin polymerization and formation of microtubule loops in growth cones over many hours, whereas Sema3A depolymerized actin filaments, attenuated microtubule dynamics, and collapsed microtubule arrays within minutes. Netrin-1 promoted rapid axon branching, often without involving the growth cone. Branches formed de novo on the axon shaft within 30 min after local application of netrin-1, which induced rapid accumulation of actin filaments in filopodia. Importantly, increased actin polymerization and microtubule dynamics were necessary for axon branching to occur. Taken together, these results show that guidance factors influence the organization and dynamics of the cytoskeleton at the growth cone and the axon shaft to promote or inhibit axon branching. Independent of axon outgrowth, axon branching in response to guidance cues can occur over different time courses by different cellular mechanisms.