The genome is folded into domains located in compartments that are either transcriptionally inert or transcriptionally permissive. Here we used genome-wide strategies to characterize domains during B cell development. Structured interaction matrix analysis showed that occupancy by the architectural protein CTCF was associated mainly with intradomain interactions, whereas sites bound by the histone acetyltransferase p300 or the transcription factors E2A or PU.1 were associated with intra- and interdomain interactions that are developmentally regulated. We identified a spectrum of genes that switched nuclear location during early B cell development. In progenitor cells, the transcriptionally inactive locus encoding early B cell factor (Ebf1) was sequestered at the nuclear lamina, which thereby preserved their multipotency. After development into the pro-B cell stage, Ebf1 and other genes switched compartments to establish new intra- and interdomain interactions associated with a B lineage-specific transcription signature.