Excitatory commissural V3 interneurons (INs) of the ventral spinal cord have been shown to balance locomotor rhythm regularity and robustness in vivo. Unfortunately, due to the scarcity of these cells in the spinal cord, in vitro studies of dissociated V3 INs have yet to be reported. In this study, we developed an induction protocol for V3 INs from mouse embryonic stem cells. The effect of the concentration of a strong sonic hedgehog (Shh) agonist (smoothened agonist [SAG]) and retinoic acid (RA) on expression of progenitor p3 and postmitotic V3 IN transcription factor markers (ie, Nkx2.2 and Sim1) was examined. Cells were differentiated toward a more ventral fate by increasing the duration of SAG exposure from 4 days in a previously established motoneuron induction protocol to 6 days. At the end of the induction period, transcription factor expression was assessed using quantitative real-time polymerase chain reaction, immunocytochemistry, in situ hybridization, and flow cytometry. Lower concentrations of RA and a longer duration of SAG exposure led to increased levels of p3 and V3 marker expression. This novel induction protocol reveals the importance of Shh signaling duration in the dorsal-ventral patterning of the neural tube, and it provides a method to obtain V3 INs for future studies to allow better understanding their role in rewiring and regeneration after spinal cord injury. © Mary Ann Liebert, Inc. 2015.