Precise spatiotemporal regulation of gene expression is critical for the development and functioning of complex, multicellular organisms. Enhancers play a fundamental role in the regulation of gene expression, but the molecular underpinnings of enhancer-mediated gene activation remain poorly understood. Many mammalian genes are dependent on the activity of multiple enhancers, which can be separated from their target genes by large genomic distances. Accurate gene regulation therefore relies on specific interactions between enhancers and their target genes in 3D nuclear space. In this review, we discuss recent insights into the mechanisms by which enhancers cooperate to regulate precise and robust gene expression levels. We also review recent progress in our understanding of the molecular drivers of specific 3D interactions between enhancers and their target genes. We conclude by discussing current models of the molecular mechanisms by which enhancers activate gene expression in their 3D chromatin context.