X chromosome inactivation (XCI) is a coordinated set of epigenetic mechanisms that leads to inactivation of the majority of genes on one of the two X chromosomes in female mammals. During XCI, gene silencing happens in three main stages: initiation, establishment and maintenance. Structural maintenance of chromosomes flexible hinge domain containing 1 (Smchd1) is an epigenetic modifier that plays an important role in XCI. Although previous studies demonstrate that Smchd1 is important in maintenance of XCI, the role of Smchd1 in establishment is not been clearly defined. Our initial immunofluorescence studies in mouse epiblast stem cells (mEpiSC) derived from E6.5 and E7.5 embryos suggest that Smchd1 is recruited to the inactive X (Xi) between E6.5 to E7.5. To further understand Smchd1’s role in XCI during this developmental window, we are performing allele-specific RNA-seq in Smchd1 deleted embryos. To understand the dynamics of Smchd1 binding to the Xi, we performed Fluorescence Recovery After Photo bleaching (FRAP) on immortalised female mouse embryonic fibroblasts (MEFs) carrying a Smchd1-GFP knock-in allele. We analysed both Smchd1 wild type and our gain of function Smchd1 mutant (Fresia) and have identified that there is a significant increase in the rate of reloading in the Fresia mutant, indicating differences in Smchd1-GFP dynamics within the nucleus. We plan to carry out similar studies in cells that carry a loss of function mutation in Smchd1. We are also using lattice light sheet microscopy to understand the reloading kinetics of Smchd1 during cell division. Finally, we performed 3D Structural illumination microscopy (3D-SIM) studies to discover possible variation in structural conformation of Smchd1 protein in Xi by tagging both ends of the protein molecule. This will help to further our understanding of the molecular mechanisms of Smchd1 that contribute to both normal gene silencing and disease.