The non-coding RNA Xist is indispensable for X chromosome inactivation. Transcriptional control of Xist gene depends on its antisense partner gene Tsix which prevents Xist up-regulation in cis. Previous studies proposed Tsix acts by regulating chromatin structure. Although histone modifications in the Xist locus during differentiation have been described in female embryonic stem (ES) cells, they remain unclear in males. Here we addressed histone modifications in the Xist locus in wild-type and Tsix-mutant male ES cells during differentiation. Their active and repressive modifications were attenuated upon differentiation, while the histone modification profile in males resembled that of females in an undifferentiated condition. These results provide implications in understanding the regulation of Xist gene, as well as other developmentally regulated genes, through chromatin structure.