The interaction between flap endonuclease 1 (FEN-1) and proliferation cell nuclear antigen (PCNA) is critical for faithful and efficient Okazaki fragment maturation. In a living cell, this interaction is probably important for PCNA to load FEN-1 to the replication fork, to coordinate the sequential functions of FEN-1 and other enzymes, and to stimulate its enzyme activity. The FEN-1/PCNA interaction is mediated by the motif (337)QGRLDDFFK(345) of FEN-1, such that an F343AF344A (FFAA) mutant cannot bind to PCNA but retains its nuclease activities. To determine the physiological roles of the FEN-1/PCNA interaction in a mammalian system, we knocked the FFAA Fen1 mutation into the Fen1 gene locus of mice. FFAA/FFAA mouse embryo fibroblasts underwent DNA replication and division at a slower pace, and FFAA/FFAA mutant embryos displayed significant defects in growth and development, particularly in the lung and blood systems. All newborn FFAA mutant pups died at birth, likely due to pulmonary hypoplasia and pancytopenia. Collectively, our data demonstrate the importance of the FEN-1/PCNA complex in DNA replication and in the embryonic development of mice.