LIM domain-containing transcription factors are required for embryonic survival and for the determination of many cell types. The combinatorial expression of the LIM homeodomain proteins Isl1, Isl2, Lhx1, and Lhx3 in subsets of developing motor neurons correlates with the future organization of these neurons into motor columns with distinct innervation targets, implying a functional role for LIM homeodomain protein combinations in the specification of neuronal identity. NLI is a widely expressed, dimeric protein that has been shown to specifically interact with the LIM domains of LIM domain-containing transcription factors. The present studies demonstrate that NLI mediates homo- and heteromeric complex formation between LIM domain transcription factors, requiring both the N-terminal dimerization and C-terminal LIM interaction domains of NLI. Although the interaction between most LIM homeodomain proteins is dependent on NLI, a direct interaction between the LIM domains of Lhx3 and the homeodomains of Isl1 and Isl2 was also observed. This interaction was disrupted by NLI, demonstrating that the conformational state of Lhx3-Isl1/Isl2 complexes is modified by NLI. Evidence indicating that NLI facilitates long range enhancer-promoter interactions suggests that NLI-dependent LIM domain transcription factor complexes are involved in communication between transcriptional control elements.