During mouse pre-implantation development, the genome undergoes a series of major epigenetic changes required for embryonic gene expression, the maintenance of totipotency, and the first differentiation events [1,2]. While many studies have established the importance of DNA methylation in epigenetic reprogramming, recent data point to a crucial role of chromatin in this process [3–5]. Yet we know very little about the role of histone modifying enzymes, chromatin remodeling factors, and histone chaperones during pre-implantation development, or in stem cells derived from early embryos [6]. A subset of identified histone chaperones and chromatin remodeling complexes can collaborate to promote nucleosome assembly in vitro and are therefore in a strategic position to control chromatin assembly and maturation during development [7,8]. Among histone chaperones, chromatin assembly factor 1 (CAF-1) is a three-subunit (p150, p60, and p48) complex which promotes histone H3 and H4 deposition onto newly synthesized DNA during replication or DNA repair [9,10]. Specifically, CAF-1 deposits the histone H3 variant H3.1 into chromatin, in a pathway coupled to DNA synthesis, whereas a second histone chaperone, HIRA, is involved in the deposition of the H3.3 variant in a DNA synthesis independent pathway [11,12]. In addition to this specific chromatin assembly activity, CAF-1 interacts with several proteins present in heterochromatin, including heterochromatin protein 1 (HP1) and MBD1, a methyl-CpG binding domain protein that recruits histone deacetylase and repressive histone methyltransferase activities [13–15]. Furthermore, p150CAF-1 is required to ensure a replication-specific pool of HP1 molecules at replication sites in pericentric heterochromatin during mid-late S phase [16]. Taken together, these data suggest roles for CAF-1 in the formation of heterochromatin and in the heritability of epigenetic traits. While the function of this evolutionary conserved histone chaperone has been studied extensively biochemically, we still lack information concerning its importance during early development in mammals and in pluripotent cells such as embryonic stem (ES) cells. Here, we have analyzed the importance of CAF-1 during early mouse development by genetic ablation and in ES cells by depletion using RNA interference (RNAi). We show that CAF-1 is essential for viability in early mouse embryos and ES cells. We provide evidence that CAF-1 is required for the spatial organization and epigenetic marking of heterochromatin domains in pluripotent embryonic cells.