Fhit gene is frequently lost or reduced in expression in various human cancers. Fhit loss initiates DNA double-strand breaks (DSBs) and subsequent genome instability. Down-regulation of thymidine kinase 1 (TK1), due to loss of Fhit, causes dNTP imbalance, resulting in spontaneous replication stress that leads to chromosomal aberrations, allele copy number variations, small insertions/deletions and single-base substitutions (SBSs). Therefore, to confirm the role of the Fhit-TK1 pathway in promoting genome stability, we asked if Fhit-deficient cells exhibit decreased levels of DNA damage upon addition of a continuous supply of thymidine, the substrate for TK1, despite the low TK1 protein expression of Fhit−/− cells. We first assessed spontaneous levels of DNA damage by quantifying nuclear γH2AX foci, marker of DSBs, by indirect immunofluorescence in early passage Fhit+/+ and −/− kidney cell lines. The Fhit−/− cells exhibited ∼2-fold increases in γH2AX positive foci vs Fhit+/+ cells. Levels of DNA damage prior to thymidine supplementation were also measured in these cells by neutral comet assay. We observed a significant elevated levels of DNA damage in Fhit−/− vs+/+ cells. Low level concentration (10 μM) thymidine supplementation suppressed DSB formation and accumulation of DNA damage in Fhit−/− cells. We also demonstrated that Fhit regulates dTTP levels and suggested that this occurs through scavenger decapping of TK1 mRNA. These results revealed that TK1 down-regulation by Fhit loss is a transient step initiating genome instability in preneoplastic lesions. The cause of Fhit-deficient DSBs: thymidine deficiency-induced replication stress, can be resolved with thymidine supplementation.