For various potential clinical applications, the use of autologous human MSCs (hMSCs) would be favorable. In vitro observations suggested that hMSCs are resistant for chemotherapeutic substances; however, no data exist on the characteristics of hMSCs from bone marrow (BM) of chemotherapeutically treated patients. Here, we analyzed the character of hMSCs derived from chemotherapy-exposed BM and the in vitro response of hMSCs to chemotherapeutic substances. Colony-forming units-fibroblast (CFU-Fs) were isolated from BM aspirates of patients after high-dose or standard chemotherapy and of donors with unaffected BM. CFU-Fs from chemotherapy-exposed and unaffected BM contained hMSCs with similar phenotype, proliferation capacity, and differentiation potential. No obvious influence of age, sex, or time since chemotherapy exposure on the presence and characteristics of hMSCs was observed. In vitro, hMSCs showed a significant resistance for cisplatin, vincristine, and etoposide compared with sensitive tumor cell lines, particularly at apoptosis-inducing doses. The phenotype and differentiation potential of hMSCs was not altered by genotoxic treatment under clinically relevant conditions in vitro. hMSCs showed an elevated threshold for cisplatin-induced apoptosis, which was characterized by a lack of caspase-9 activity in apoptotic cells. In vitro exposure of hMSCs to cisplatin, vincristine, and etoposide resulted in an increased p53 expression, independent of apoptosis induction. We conclude that hMSCs can be isolated from chemotherapy-exposed BM in sufficient number and quality for potential clinical applications in chemotherapeutically treated patients. Our data suggest that an elevated apoptotic threshold contributes not only to the persistence of hMSCs in the BM after chemotherapy but also to their lifelong presence in the adult BM.