During vertebrate development, H3 histone variants H3.1/2 and H3.3 cannot substitute for each other. It has been a longstanding puzzle, whether this is due to unique properties of individual variants or if it simply reflects their different incorporation pathways (DNA-synthesis dependent or independent). In Xenopus we demonstrated that only the presence of serine at the position 31 (S31) in H3.3 was essential at gastrulation, independent of the incorporation pathway. Moreover, the phosphorylation at S31 promotes acetylation at lysine 27, thus contributing to an open chromatin state. These findings illustrate how a unique residue in H3.3 allows the establishment and the maintenance of particular chromatin states, which are important during cell cycle and development.