Regulation of directed axon guidance and branching during development is essential for the generation of neuronal networks. However, the molecular mechanisms that underlie interstitial (or collateral) axon branching in the mammalian brain remain unresolved. Here, we investigate interstitial axon branching in vivo using an approach for precise labeling of layer 2/3 callosal projection neurons (CPNs). This method allows for quantitative analysis of axonal morphology at high acuity and also manipulation of gene expression in well-defined temporal windows. We find that the GSK3β serine/threonine kinase promotes interstitial axon branching in layer 2/3 CPNs by releasing MAP1B-mediated inhibition of axon branching. Further, we find that the tubulin tyrosination cycle is a key downstream component of GSK3β/MAP1B signaling. These data suggest a cell-autonomous molecular regulation of cortical neuron axon morphology, in which GSK3β can release a MAP1B-mediated brake on interstitial axon branching upstream of the posttranslational tubulin code. Synopsis: In the mammalian brain cortex, correct layer-specific interstitial (collateral) axon branching is required for generating functional circuitry. Here, precise gene manipulation and defined neuronal labeling in the murine neocortex identifies a role for microtubule-binding protein MAP1B in inhibiting axon interstitial branching. GSK3β activation induces excessive interstitial axon branching in excitatory cortical neurons. MAP1B inhibits axon branching downstream of GSK3β. GSK3β phosphorylates MAP1B to release its inhibitory effect on axon branching. GSK3β and MAP1B regulate interstitial axon branching through modulation of tubulin tyrosination. Microtubule-binding protein MAP1B Regulates Interstitial Axon Branching of Cortical Neurons via the Tubulin Tyrosination Cycle. [ABSTRACT FROM AUTHOR]