Histone H3.3, a variant of H3, is encoded by 2 genes: H3F3A and H3F3B. H3.3 has a Serine at residue 31 which is evolutionarily conserved and is known to be phosphorylated during metaphase. The enrichment of H3.3S31ph has been observed at heterochromatic repeats including the telomeres and pericentric satellite DNA. The presence of H3.3S31ph at these repeats is dependent on ATRX-mediated H3.3 loading. To date, little is known about the function and regulation of H3.3S31ph. In this study, we aim to define the function of H3.3S31ph, in particular, its role in telomeric chromatin assembly. We generated Cre-mediated H3.3 knockout mouse embryonic stem cell (ESC) lines expressing phospho-mimic (H3.3S31E) and phospho-null (H3.3S31A) mutants of H3.3 and studied its effects on chromatin.