The dynamics of chromatin structure are tightly regulated by multiple epigenetic mechanisms such as histone modifications and incorporation of histone variants. In the current work, differentiation of an embryonal carcinoma cell line, NT2, was induced by retinoic acid, and total histone proteins were compared throughout this process. The results showed a significant change in expression level of a variant of H1 histone named H1x. Chromatin immunoprecipitation coupled with real-time PCR analysis demonstrated a preferential incorporation of this protein in the regulatory region of Nanog, a marker gene of stemness that is significantly suppressed in differentiated cells. This finding reveals a dynamic role of H1x in differentiation, and implies a repressive role for this histone variant.