Organ transplantation depends on the lifelong use of immunosuppressive drugs to combat graft rejection, but prolonged immunosuppression is associated with opportunistic infections and malignancy. To avoid this problem, donor-derived haematopoietic stem cells (HSCs) could be transplanted into the recipient prior to the organ transplant, to create a chimeric immune system that is tolerant to both host cells and donor graft. However, in aged recipients, reduced uptake of transplanted HSCs in the aged bone marrow is compounded by poor immune recovery due to age-related atrophy of the thymus. We therefore investigated minimal bone marrow conditioning regimes that would mitigate the damage to the thymus and allow for long-term transplant acceptance in aged mice. In contrast to low-dose irradiation and cyclophosphamide, busulfan caused only minimal damage to the thymus and facilitated highly efficient engraftment of donor HSCs. Chimeric mice accepted donor skin grafts and rejected third-party grafts, indicating immune competence. In this model, peripheral regulation operated in the absence of a thymus, and tolerance to donor graft could be induced despite thymic atrophy and immunosenescence. We further established a transplantation threshold for donor HSC dose and found that mobilisation of recipient HSCs could increase the sensitivity of aged HSCs to busulfan treatment. Our findings are especially relevant in the clinic for the application of HSC transplantation and tolerance induction in older patients who make up the majority of transplant recipients.