During development, navigating axons have to decide whether or not to cross the midline in order to project to their specific cellular targets. We have recently demonstrated in mice that a single homeodomain transcription factor Dbx1, expressed by a subset of progenitor cells in the dorsal midbrain, has an essential function in assigning midline-crossing identity to midbrain commissural neurons, thereby contributing crucially to the establishment of the wiring laterality in the developing brain (Inamata & Shirasaki, Developmemt 2014). We also provided evidence that Dbx1 initiates a downstream molecular program that controls the expression of Robo3, an essential regulator of midline crossing, on commissural neurons, while repressing the ipsilateral neuron genetic program in parallel. Because the Dbx1 is a transcriptional factor operating at the progenitor stage prior to axonogenesis, a critical issue still remained regarding the downstream molecular programs within the Dbx1-triggered transcriptional cascade acting at the postmitotic stage. In the present study, to address this, we focused our analysis on postmitotically expressed transcription factors in the dorsal midbrain. We show that the homeodomain transcription factor Evx2 is expressed selectively by Robo3-positive commissural neurons. As predicted, misexpression of Dbx1 using in vivo electroporation in mice dramatically induces an ectopic expression of Evx2, misexpression of which further elicits Robo3 expression. Because Dbx1 was previously shown to suppress the expression of POU factor Brn3a (an ipsilateral neuron marker), we then asked whether Evx2 misexpression represses the expression of Brn3a. In addition, we also considered the significance of a preferential expression of Brn3a in ipsilateral neurons. Our results show that Evx2 is one of the critical downstream transcription factors within the Dbx1-triggered cascade, which is responsible for the induction of Robo3 expression at the postmitotic stage. Furthermore, it is suggested that the suppression of Brn3a expression in commissural neurons is critical for commissural neuron specification in order to restrain the potential to express conflicting genetic programs employed in ipsilateral neurons.