Locomotion is controlled by Central Pattern Generators that elicit rhythmic locomotor movements of the limbs. These circuits located in the ventral part of the spinal cord are composed of motor neurons and interneurons. In the developing spinal cord, transcriptional regulators, including Lim-Homeodomain (Lim-HD) transcription factors, cooperate to segregate motor neurons and different interneuron subsets with distinct molecular, morphological, and functional characteristics. Based on their high sequence homology and their similar distribution, Lim-HD paralogs of the Lim3 group (Lhx3 and Lhx4) are considered to contribute similarly to this process. However, the specific and individual contribution of these paralogs remains unclear. Here, we compared the activity of these factors and their interactions for motor neuron and V2 interneuron differentiation. We provide evidence that, similarly to Lhx3, Lhx4 can form multiproteic complexes with the nuclear Lim interactor NLI and the Lim-HD proteins Isl1 and Isl2. We show in chicken embryos that Lhx4, like Lhx3, can stimulate motor neuron differentiation. However, Lhx4 is more potent than Lhx3 to activate the V2-promoting TeRE enhancer and to stimulate the differentiation of V2a interneurons. In addition, the loss of Lhx4 in mouse embryos does not alter motor neuron development but results in a reduction in the differentiation of V2a interneurons. Thus, Lhx4 could be the most efficient Lim-HD factor to promote V2a interneuron differentiation during spinal cord development and should be considered for in vitro production of this spinal neuronal population. These data further complement the current model of V2 fate acquisition in the developing spinal cord.