AbstractWe report that preexisting (old) and newly synthesized (new) histones H3 and H4 are asymmetrically partitioned during the division ofDrosophilaintestinal stem cells (ISCs). Furthermore, the inheritance patterns of old and new H3 and H4 in postmitotic cell pairs correlate with distinct expression patterns of Delta, an important cell fate gene. To understand the biological significance of this phenomenon, we expressed a mutant H3T3A to compromise asymmetric histone inheritance. Under this condition, we observe an increase in Delta‐symmetric cell pairs and overpopulated ISC‐like, Delta‐positive cells. Single‐cell RNA‐seq assays further indicate that H3T3A expression compromises ISC differentiation. Together, our results indicate that asymmetric histone inheritance potentially contributes to establishing distinct cell identities in a somatic stem cell lineage, consistent with previous findings inDrosophilamale germline stem cells.