Faithful repair of chromosomal double-strand breaks (DSBs) is crucial to genome stability and normal cell survival and proliferation. During normal metabolic processes and especially following exposure to DNA damaging agents including irradiation, alkylating agents, and inhibitors of topoisomerase II such as etoposide chromosomal DSBs occur. More recently, researchers have investigated the potential of DSBs from exposure to dietary and environmental compounds, including bioflavonoids. Bioflavonoids are found in fruits, tea, coffee, wine, soy, and cocoa, as well as anthraquinone laxatives, podophyllin resins, flame retardants, quinolone antibiotics, pesticides, and phenolic compounds. Previous studies demonstrated that several bioflavonoids induce DNA DSBs, including cleavage within the MLL gene leading to chromosomal translocations. Importantly, the cleavage sites colocalize with cleavage sites induced by chemotherapeutic agents such as etoposide. This research focuses on the mechanisms of DNA damage signaling and the DSB repair pathways preferentially triggered following cell exposure to two specific bioflavonoids, i.e., genistein and quercetin. The hypothesis tested is that genistein and quercetin induce DNA DSBs in the MLL and AF9 genes, promote canonical/specific DNA damage responses and specifically suppress classical nonhomologous end-joining (C-NHEJ) repair but stimulate error-prone alternative end-joining (Alt-EJ) repair events that favor chromosomal translocations. Additionally, the hypothesis that genistein and quercetin mechanistically impact DNA repair pathways by affecting post-translational modifications of DNA repair proteins is tested. To address these hypotheses, parental embryonic stem cells as well as CRISPR XLF (-) XRCC4 (-) cells were treated with increasing doses of genistein and quercetin and the expression of C-NHEJ protein DNA-PKcs, XLF, XRCC4, and Alt-EJ protein CtIP, Pol θ determined. Additionally, the phosphorylation status of DNA-PKcs was defined. Results indicate that cells treated with genistein and quercetin consistently favor the Alt-EJ DNA repair pathway. Overall these findings indicate genistein and quercetin can initiate DNA damage, influence DSB repair pathway choice and affects post-translational modifications of DNA repair genes. This research significantly broadens our knowledge about the potential for supplemental dietary compounds to induce DNA damage and to influence subsequent DNA repair pathway choice.