FLASH has been shown to be required for S phase progression and to interact with a nuclear protein, ataxia-telangiectasia locus (NPAT), a component of Cajal bodies in the nucleus and an activator of histone transcription. We investigated the role of human FLASH by using an inducible FLASH knockdown system in the presence or absence of various mutant forms of mouse FLASH. While carboxyl-terminal deletion mutants of FLASH, which do not interact with NPAT, can support S phase progression, its amino-terminal deletion mutants, which are unable to self associate, cannot support S phase progression, replication-dependent histone transcription, or the formation of Cajal bodies. Furthermore, FLASH was shown to be associated with arsenite resistance protein 2 (ARS2) through its central region, which is composed of only 13 amino acids. The expression of ARS2 and the interaction between FLASH and ARS2 are required for S phase progression. Taking these results together, FLASH functions in S phase progression through interaction with ARS2.