The histone variant macroH2A occupies large repressive domains throughout the genome; however, mechanisms underlying its precise deposition remain poorly understood. Here, we characterize de novo chromatin deposition of macroH2A2 using temporal genomic profiling in murine-derived fibroblasts devoid of all macroH2A isoforms. We find that macroH2A2 is first pervasively deposited genome wide at both steady-state domains and adjacent transcribed regions, the latter of which are subsequently pruned, establishing mature macroH2A2 domains. Pruning of macroH2A2 can be counteracted by chemical inhibition of transcription. Further, locus-specific transcriptional manipulation reveals that gene activation depletes pre-existing macroH2A2, while silencing triggers ectopic macroH2A2 accumulation. We demonstrate that the FACT (facilitates chromatin transcription) complex is required for macroH2A2 pruning within transcribed chromatin. Taken together, we have identified active chromatin as a boundary for macroH2A domains through a transcription-associated 'pruning' mechanism that establishes and maintains the faithful genomic localization of macroH2A variants.