DNA damaging agents are widely used in treatment of hematogical malignancies and solid tumors. While effects on hematopoietic stem cells have been characterized, less is known about the DNA damage response in human mesenchymal stem cells (hMSCs) in the bone marrow stroma, progenitors of osteoblasts, chondrocytes and adipocytes. To elucidate the response of undifferentiated hMSCs to gamma-irradiation and cisplatin, key DNA damage responses have been characterized in hMSCs from normal adult donors. Cisplatin and gamma-irradiation activated the DNA damage response in hMSCs, including induction of p53 and p21, and activation of PI3 kinase-related protein kinase (PIKK)-dependent phosphorylation of histone H2AX on serine 139, and replication protein A2 on serine4/serine8. Chemical inhibition of ATM or DNA-PK reduced DNA damage-induced phosphorylation of H2AX, indicating a role for both PIKKs in the response of hMSCs to DNA damage. Consistent with repair of DNA strand breaks, gamma H2AX staining decreased by 24 h following gamma-irradiation. gamma-irradiation arrested hMSCs in the G 1 phase of the cell cycle, while cisplatin induced S-phase arrest, mediated in part by the ATR/Chk1 checkpoint pathway. In hMSCs isolated from a chronic lymphocytic leukemia (CLL) patient, p53 and p21 were induced by cisplatin and gamma-irradiation, while RPA2 was phosphorylated on serine4/8 in particular following cisplatin. Compared with peripheral blood lymphocytes or the leukemia cell line K562, both normal hMSCs and CLL-derived hMSCs were more resistant to cisplatin and gamma-irradiation. These results provide insights into key pathways mediating the response of bone marrow-derived hMSCs to DNA damaging agents used in cancer treatment.