The closely related replicative H3 and non-replicative H3.3 variants show specific requirement during development in vertebrates. Whether it involves distinct mode of deposition or unique roles once incorporated into chromatin remains unclear. To disentangle the two aspects, we took advantage of the Xenopus early development combined with chromatin assays. We systematically mutated H3.3 at each four residues that differ from H3.2 and tested their ability to rescue developmental defects due to endogenous H3.3 depletion. Surprisingly, all H3.3 mutated variants functionally complemented endogenous H3.3, regardless of their incorporation pathways, except for one residue, the serine at position 31. The phosphorylation at this unique residue occurs onto chromatin with a peak in late mitosis, and depends on the networks of cell cycle kinases. Notably, while the alanine substitution failed to rescue H3.3 depletion, a phosphomimic residue sufficed. Based on proteomics studies with histone peptides and Xenopus extracts, we find that the phosphomimic histone mutant attracts transcription related factors. Furthermore, we evidence a crosstalk whereby phosphorylation on H3.3S31 favors H3.3K27ac. At gastrulation, we conclude that the critical importance of the H3.3S31 residue is independent of the variant incorporation pathway. It rather reflects a signaling role engaging key binding partners and crosstalks on neighboring amino acids. We discuss how this single evolutionary conserved residue conveys both in interphase and mitosis unique properties for this variant in vertebrates during cell cycle and cell fate commitment.