In the zebrafish spinal cord, two classes of neurons develop from the lateral floor plate: Kolmer-Agduhr′ (KA′) and V3 interneurons. We show here that the differentiation of the correct number of KA′ cells depends on the activity of the homeobox transcription factor Nkx2.9. This factor acts in concert with Nkx2.2a and Nkx2.2b. These factors are also required for the expression of the zinc-finger transcription factor Gata2 in the lateral floor plate. In turn, Gata2 is necessary for expression of the basic helix-loop-helix transcription factor Tal2 that acts upstream of the GABA-synthesizing enzyme glutamic acid decarboxylase 67 gene (gad67) in KA′ cells. Expression of the transcription factor Sim1, which marks the V3 interneurons in the lateral floor plate, depends also on the three Nkx2 factors. sim1 expression does not require, however, gata2 and tal2. KA′ cells of the lateral floor plate and the KA′ cells located more dorsally in the spinal cord share expression of transcription factors. The functional connections between the different regulatory genes, however, differ in the two GABAergic cell types: although gata2 and tal2 are expressed in KA′ cells, they are dispensable for gad67 expression in these cells. Instead, olig2 and gata3 are required for the differentiation of gad67-expressing KA′ cells. This suggests that the layout of regulatory networks is crucially dependent on the lineage that differs between KA′ and KA′ cells.