Precise gene expression patterns during mammalian development are controlled by regulatory elements in the non-coding genome. Active enhancer elements interact with gene promoters within Topologically Associating Domains (TADs) 1–3 . However, the precise relationships between chromatin accessibility, nuclear architecture and gene activation are not completely understood. Here, we present Tiled-C, a new Chromosome Conformation Capture (3C) technology 4 , which allows for the generation of high-resolution contact matrices of loci of interest at unprecedented depth, and which can be optimized for as few as 2,000 cells of input material. We have used this approach to study the chromatin architecture of the mouse α -globin locus through in vivo erythroid differentiation. Integrated analysis of matched chromatin accessibility and single-cell expression data shows that the α -globin locus lies within a pre-existing TAD, which is established prior to activation of the domain. During differentiation, this TAD undergoes further sub-compartmentalization as regulatory elements gradually become accessible and specific interactions between enhancers and promoters are formed. As these chromatin changes develop, gene expression is progressively upregulated. Our findings demonstrate that chromatin architecture and gene activation are tightly linked during development and provide insights into the distinct mechanisms contributing to the establishment of tissue-specific chromatin structures.