Bone sarcomas, such as osteosarcoma (OS) or Ewing sarcoma (ES), frequently arise in the intramedullary region of long bones. Patients affected by bone sarcomas are treated with preoperative aggressive chemotherapy immediately after diagnosis. After chemotherapy, patients undergo surgery that frequently entails the excision of wide bone segments. If a large segment of the bone is lost (defined as a critical defect) the patient undergoes bone reconstruction. Because bone marrow derived stromal stem cells (SSC) offer great promise for cell-based regenerative medicine in bone reconstruction, we investigated whether SSC could be isolated from chemotherapy-treated bone sarcoma patients. We also investigated whether chemotherapy modified SSC differentiation capability. We studied 9 SSC derived from OS and ES patients that had undergone chemotherapy and 5 SSC derived from bone sarcoma patients that had not undergone chemotherapy. SSC could be obtained from all the patients analyzed regardless of whether the patients received chemotherapy or not. Our results also showed that post-chemotherapy SSC can be cultured and expanded ex vivo, these cells retained the ability to differentiate toward the osteogenic lineage in vitro. In conclusion, our results support that SSC cells can be obtained from bone sarcoma patients that undergo chemotherapy and suggest that SSC can be used for cell-based bone reconstruction techniques in bone sarcoma patients treated with preoperative chemotherapy.