A large fraction of the mammalian genome is organized into inactive chromosomal domains along the nuclear lamina. The mechanism by which these lamina associated domains (LADs) are established remains to be elucidated. Using genomic repositioning assays, we show that LADs, spanning the developmentally regulated IgH and Cyp3a loci contain discrete DNA regions that associate chromatin with the nuclear lamina and repress gene activity in fibroblasts. Lamina interaction is established during mitosis and likely involves the localized recruitment of Lamin B during late anaphase. Fine-scale mapping of LADs reveals numerous lamina-associating sequences (LASs), which are enriched for a GAGA motif. This repeated motif directs lamina association and is bound by the transcriptional repressor cKrox, in a complex with HDAC3 and Lap2β. Knockdown of cKrox or HDAC3 results in dissociation of LASs/LADs from the nuclear lamina. These results reveal a mechanism that couples nuclear compartmentalization of chromatin domains with the control of gene activity. Display omitted ► Lamina-associating DNA sequences (LASs) direct chromatin to the nuclear lamina ► In mammalian cells, LASs are bound by the transcriptional repressor cKrox ► cKrox recruits DNA to the lamina via HDAC3 and lamina-associated protein Lap2β ► Lamina interaction is established during mitosis, likely through recruitment of Lamin B Binding of a transcriptional repressor complex to DNA sequences enriched in GAGA motifs directs the association of specific chromosome domains to the nuclear lamina during mitosis. These lamin-associated sequences thus couple nuclear compartmentalization of chromatin domains with gene silencing.