During development, cortical interneurons generated from the ventral telencephalon migrate tangentially into the dorsal telencephalon. Although Achaete-scute family bHLH transcription factor 1 (Ascl1) plays important roles in the developing telencephalon, whether Ascl1 regulates tangential migration remains unclear. Here, we found that Ascl1 promoted tangential migration along the ventricular zone/subventricular zone (VZ/SVZ) and intermediate zone (IZ) of the dorsal telencephalon. Distal-less homeobox 2 (Dlx2) acted downstream of Ascl1 in promoting tangential migration along the VZ/SVZ but not IZ. We further identified Eph receptor B2 (Ephb2) as a direct target of Ascl1. Knockdown of EphB2 disrupted the separation of the VZ/SVZ and IZ migratory routes. Ephrin-A5, a ligand of EphB2, was sufficient to repel both Ascl1-expressing cells in vitro and tangentially migrating cortical interneurons in vivo. Together, our results demonstrate that Ascl1 induces expression of Dlx2 and Ephb2 to maintain distinct tangential migratory routes in the dorsal telencephalon.The cerebral cortex is important for executing high-order brain functions, such as sensory perception, motor control and cognition. Proper functions of the cerebral cortex require coordination between excitatory (glutamatergic) projection neurons and inhibitory (GABAergic) interneurons. Many neurological and psychological disorders, such as epilepsy, autism, schizophrenia, and Alzheimer's disease, may be resulted from an imbalance of excitatory and inhibitory neuronal activities or the dysfunction of interneurons 1,2 . Interestingly, glutamatergic neurons and GABAergic interneurons are produced from different regions of the developing brain. Glutamatergic neurons are generated from neural progenitors in the dorsal telencephalon, which develops into the cerebral cortex. GABAergic cortical interneurons are remotely derived from neural progenitors in the ventral telencephalon, which develops into the striatum and globus pallidus 3 . Around 85% of cortical interneurons are generated from the medial ganglionic eminence (MGE) and the rest are generated from the caudal ganglionic eminence (CGE) 4,5 . Once generated in the ventricular zone (VZ), glutamatergic neurons and GABAergic interneurons migrate toward final destinations through distinct paths. Glutamatergic neurons undergo radial migration to reach the cortical plate (CP) 6 . Interneurons take a long tangentially migratory path from the ventral telencephalon toward the striatum or the cerebral cortex 3 . When cortical interneurons reach the dorsal telencephalon, they continue migrating tangentially through three migratory routes: the marginal zone (MZ), intermediate zone (IZ) or ventricular/subventricular zone (VZ/SVZ) of the dorsal telencephalon 7,8 . Upon arrival at destined region, interneurons switch to migrate radially until they reach specific layers of the cerebral cortex, where they integrate into local neuronal circuits. Since cortical interneurons go through such a complicated migratory path, it is impo...