Long-range interactions between distant regulatory elements, such as enhancers, and their target genes underlie the specificity of gene expression in many developmentally regulated gene families. NLI/Ldb1, a widely expressed nuclear factor, is a potential mediator of long-range interactions. Here, we show that NLI/Ldb1 and erythroid-binding partners GATA-1/SCL/LMO2 bind in vivo to the β-globin locus control region (LCR). The C-terminal LIM interaction domain of NLI is required for formation of the complex on chromatin. Loss of the LIM domain converts NLI into a dominant-negative inhibitor of globin gene expression, and knockdown of NLI by using shRNA results in failure to activate β-globin expression. Kinetic studies reveal that the NLI/GATA-1/SCL/LMO2 complex is detected at the β-globin promoter coincident with RNA Pol II recruitment, β-globin transcription, and chromatin loop formation during erythroid differentiation, providing evidence that NLI facilitates long-range gene activation.