Meristems retain the ability to divide throughout the life cycle of plants, which can last for over 1000 years in some species. Furthermore, the germline is not laid down early during embryogenesis but originates from the meristematic cells relatively late during development. Thus, accurate cell cycle regulation is of utmost importance to avoid the accumulation of mutations during vegetative growth and reproduction. The Arabidopsis thaliana genome encodes two homologs of the replication licensing factor CDC10 Target1 (CDT1), and overexpression of CDT1a stimulates DNA replication. Here, we have investigated the respective functions of Arabidopsis CDT1a and CDT1b. We show that CDT1 proteins have partially redundant functions during gametophyte development and are required for the maintenance of genome integrity. Furthermore, CDT1-RNAi plants show endogenous DNA stress, are more tolerant than the wild type to DNA-damaging agents, and show constitutive induction of genes involved in DNA repair. This DNA stress response may be a direct consequence of reduced CDT1 accumulation on DNA repair or may relate to the ability of CDT1 proteins to form complexes with DNA polymerase ε, which functions in DNA replication and in DNA stress checkpoint activation. Taken together, our results provide evidence for a crucial role of Arabidopsis CDT1 proteins in genome stability.