Eukaryotic cells have a layer of heterochromatin at the nuclear periphery. To investigate mechanisms regulating chromatin distribution, we analyzed heterochromatin organization in different tissues and species, including mice with mutations in the lamin B receptor (Lbr) and lamin A (Lmna) genes that encode nuclear envelope (NE) proteins. We identified LBR- and lamin-A/C-dependent mechanisms tethering heterochromatin to the NE. The two tethers are sequentially used during cellular differentiation and development: first the LBR- and then the lamin-A/C-dependent tether. The absence of both LBR and lamin A/C leads to loss of peripheral heterochromatin and an inverted architecture with heterochromatin localizing to the nuclear interior. Myoblast transcriptome analyses indicated that selective disruption of the LBR- or lamin-A-dependent heterochromatin tethers have opposite effects on muscle gene expression, either increasing or decreasing, respectively. These results show how changes in NE composition contribute to regulating heterochromatin positioning, gene expression, and cellular differentiation during development. Display omitted ► LBR- and lamin-A/C-dependent tethers maintain peripheral heterochromatin ► In their absence, all heterochromatin clusters in the nuclear interior ► During cellular differentiation, the LBR tether precedes the lamin A/C tether ► Lamin A/C promotes, whereas LBR delays myogenic differentiation The lamin B receptor and lamin A/C have sequential roles in the tethering of peripheral heterochromatin during cellular differentiation, corresponding to their differential effects on heterochromatin positioning and transcription of tissue-specific genes.