It has been long speculated that mammalian Rev3 plays an important, yet unknown role(s) during mammalian development, as deletion of Rev3 causes embryonic lethality in mice, whereas no other translesion DNA synthesis polymerases studied to date are required for mouse embryo development. Here, we report that both subunits of Polζ (Rev3 and Rev7) show an unexpected increase in expression during G(2)/M phase, but they localize independently in mitotic cells. Experimental depletion of Rev3 results in a significant increase in anaphase bridges, chromosomal breaks/gaps and common fragile site (CFS) expression, whereas Rev7 depletion primarily causes lagging chromosome defect with no sign of CFS expression. The genomic instability induced by Rev3 depletion seems to be related to replication stress, as it is further enhanced on aphidicolin treatment and results in increased metaphase-specific Fanconi anemia complementation group D type 2 (FANCD2) foci formation, as well as FANCD2-positive anaphase bridges. Indeed, a long-term depletion of Rev3 in cultured human cells results in massive genomic instability and severe cell cycle arrest. The aforementioned observations collectively support a notion that Rev3 is required for the efficient replication of CFSs during G(2)/M phase, and that the resulting fragile site instability in Rev3 knockout mice may trigger cell death during embryonic development.