Updated publication reference for PubMed record(s): 28366643. Histone replacement by transition proteins (TPs) and protamines (Prms) constitutes an essential step for the successful production of functional male gametes, yet nothing is known on the underlying functional interplay between histones, TPs and Prms. Here, by studying spermatogenesis in the absence of H2A.L.2, a spermatid-specific histone variant, we identify a new step, which is essential for the transformation of nucleosomes to nucleoprotamines. H2A.L.2, synthesized at the same time as TPs, enables the invasion of nucleosomes by TPs, which themselves drive the recruitment and processing of Prms. H2A.L.2 incorporation initiates a series of successive transitional states that ultimately shift to a more stable state characterized by nucleoprotamine assembly and histone-TP displacement, leading to the generation of mature spermatozoa. Hence, the current view of histone-to-nucleoprotein transition should be revisited and include a new step with H2A.L.2 assembly prior to the action of TPs and Prms.