The chromatin assembly factor 1 (CAF-1) is a three-subunit protein complex that functions in chaperoning histone deposition. The smallest subunit of Drosophila CAF-1, dCAF-1-p55, together with the other two subunits dCAF-1-p180 and dCAF-1-p105, participates in the deposition of histone H3 and H4 onto the newly synthesized DNA. In addition to the CAF-1 complex, dCAF-1-p55 is also found in several other complexes such as NURF, PRC2 and Sin3-HDAC1 complex, suggesting that it plays multiple roles other than a histone chaperone. Here we report a new allele of dCAF-1-p55 generated by gene targeting. We show that dCAF-1-p55 loss-of-function led to developmental delay and lethality. Further investigations demonstrated that dCAF-1-p55 deficient cells exhibited defects in metaphase chromosome condensation, sister chromatids association and anaphase chromosomes segregation, which are premonitory signs of chromosome instability (CIN). Taken together, these results indicate that dCAF-1-p55 plays a role in the maintenance of chromosomal stability, likely to protect the cells from CIN and the proneness to cancer.