The vertebrate Dlx gene family consists of multiple convergently transcribed bigene clusters and encodes a group of homeodomain-containing transcription factors crucial for the development of forebrain, branchial arches, sensory organs and limbs. At least four cis-regulatory elements (CREs) are responsible for Dlx expression in the forebrain: URE2 and I12b in the Dlx1/Dlx2 (zebrafish dlx1a/dlx2a) locus, and, I56i and I56ii in the Dlx5/Dlx6 (zebrafish dlx5a/dlx6a) locus. Here, we first show that unlike the other three enhancers, mouse I56ii CRE targets a group of GABAergic projection neurons expressing striatal markers Meis2 and Islet1. Meis2 and Islet1 proteins can activate reporter gene transcription via the I56ii CRE, suggesting that they may be potential upstream regulators of Dlx genes in vivo. To determine whether there exists a dlx-mediated regulatory pathway during zebrafish GABAergic neuron formation, we establish two independent lines of transgenic fish in which the GFP reporter gene is controlled by a 1.4kb dlx5a/dlx6a intergenic sequence (encompassing zebrafish I56i and I56ii) and a 1.1kb fragment containing only I56i CRE, respectively. Our observations reveal that dlx5a/dlx6a regulatory elements exhibit a fairly specific activity in the zebrafish forebrain and may be essential for GABAergic neuron generation, while I56i and I56ii are likely to play distinct roles in modulating this process in different subpopulations of cells. Disruption of dlx1a/dlx2a or dlx5a/dlx6a function leads to a marked decrease of enhancer activity in the diencephalon and midbrain as well as a comparatively lesser extent of reduction in the telencephalon. In order to define the specific contribution of various individual CREs to overall Dlx regulation, we also generate a mutant mouse model in which I12b CRE is selectively deleted. Despite that mice homozygous for I12b loss develop normally and harbor no overt morphological defects in the forebrain, targeted deletion of this enhancer results in a significant reduction of Dlx1/Dlx2 transcript levels and seemingly perturbs cell proliferation in the subpallial telencephalon, particularly in the ventricular and subventricular zones of ganglionic eminences. Taken together, these data illustrate a complex and dynamic Dlx regulation in the early developing forebrain through the implications of multiple Dlx CREs with overlapping and diverse functions.