The l3mbtl1 gene is located on the long arm of chromosome 20 (q12), within a region commonly deleted in several myeloid malignancies. L3MBTL1 is a human homolog of the Drosophila polycomb L(3)MBT tumor suppressor protein and thus a candidate tumor suppressor in del(20q12) myeloid disorders. We used the loss-of-function approach to explore the possible tumor suppressive mechanism of L3MBTL1 and found that depletion of L3MBTL1 from human cells causes replicative stress, DNA breaks, activation of the DNA damage response, and genomic instability. L3MBTL1 interacts with Cdc45, MCM2-7 and PCNA, components of the DNA replication machinery, and is required for normal replication fork progression, suggesting that L3MBTL1 causes DNA damage, at least in part, by perturbing DNA replication. An activated DNA damage response and genomic instability are common features in tumorigenesis and a consequence of overexpression of many oncogenes. We propose that the loss of L3MBTL1 contributes to the development of 20q⁻ hematopoietic malignancies by inducing replicative stress, DNA damage, and genomic instability.