LIM-homeodomain (LIM-HD) transcription factors act as key developmental regulators, both through their ability to bind DNA through homeodomain:DNA interactions, and through their ability to form larger complexes through protein:protein interactions. The LIM-HD proteins Islet-1 (Isl1) and LIM homeobox protein 3 (Lhx3) have been implicated in the development of many tissues. The best studied of these is their role in cell fate determination in the developing central nervous system. These proteins, along with the protein cofactor LIM domain binding protein 1 (Ldb1) interact via LIM:LID interactions, to form cell-specific transcriptional complexes that target different genes than are targeted by either protein alone. It is not known if the homeodomains target these different sites solely because of the LIM-LID interactions or if the homeodomains bind cooperatively to DNA. These interactions were investigated, through comparative electrophoretic mobility shift assay (EMSA) experiments and quantitative microscale thermophoresis (MST) experiments. Data indicated no apparent cooperativity of binding between the two isolated homeodomains. However, by comparing the DNA-binding ability of protein constructs in which the homeodomains from each protein were tethered, either with no additional interaction domains, or in the presence of the LIM and LID domains, it was shown that the latter bound DNA much more effectively. Thus the protein interaction domains involved in the formation of the Lhx3:Isl1:Ldb1 transcriptional complex may play a larger role in DNA binding than previously thought, and thereby help to define cell specific transcriptional complexes.