Mammalian embryos undergo dramatic membrane reorganisation as they develop from a 1-cell zygote to a multicellular blastocyst. In addition to structural roles, cell membrane integrity is essential for signalling via receptors associated with lipid raft microdomains. Lipid rafts undergo disintegration in response to cellular cholesterol depletion and this sensitivity can be exploited to identify essential roles of lipid microdomains. Oocytes are cholesterol-laden cells and we sought to determine whether oocyte cholesterol stores are important for establishing membrane structure and functional lipid rafts during embryogenesis. Cholesterol deposition in oocytes and embryos was detected by staining with filipin. Lipid rafts were visualised using the marker ganglioside GM1, and concentrated on the membrane of oocytes and trophectoderm (TE) and inner cell mass (ICM) cells of blastocysts. Next, ovulated cumulus-oocyte complexes (COCs) were treated for 30 minutes with methyl-b-cyclodextrin (MbCD), which extracts cholesterol from the cell membrane, followed by in-vitro fertilisation (IVF) and assessments of embryo development. An effective dose of MbCD was established which significantly decreased cholesterol in oocytes (p<0.05) yet did not affect viability. Cholesterol depletion also significantly impaired cleavage rate and blastocyst development following IVF (p<0.05). The embryos derived from cholesterol-depleted oocytes were examined for membrane and lipid raft localisation and markers of embryo health, including mitochondrial membrane potential (MMP), ROS levels and allocation of cells to TE and ICM. MMP in blastocysts was not affected by oocyte cholesterol depletion. However, embryos derived from oocytes depleted of cholesterol exhibited a clear disruption to membrane structure. Specifically the tight junction permeability seals between the TE important for blastocyst formation were not visible. Oocyte cholesterol depletion also disrupted lipid raft distribution in the mouse embryos (p<0.05). These results demonstrate the importance of oocyte cholesterol stores for lipid raft formation and function, and the essential role of membrane organisation for preimplantation embryogenesis.