In vertebrates, spinal commissural axons project along a transverse path toward and across the floor plate (FP). Post-crossing commissural axons alter their responsiveness to FP-associated guidance cues and turn to project longitudinally in a fasciculated manner prior to extending away from the midline. The upregulation of the neural cell adhesion molecule L1 on crossed commissural axon segments has been proposed to facilitate pathfinding on the contralateral side of the FP. To explore this possibility in vivo, we used Math1 regulatory sequences to target L1 to commissural axons before they cross the ventral midline. L1 mis-expression did not alter the distribution of commissural axon-associated markers or the ventral extension of commissural axons toward the midline. However, commissural axons often stalled or inappropriately projected into the longitudinal plane at the ipsilateral FP margin. These observations suggest that L1-mediated pathfinding decisions are normally delayed until axons have crossed the ventral midline (VM).