Hematopoietic stem cell (HSC) engraftment has been achieved using single-cell transplantation of prospectively highly purified adult HSC populations. However, bulk transplants are still performed when assessing the HSC potential of early embryonic hematopoietic tissues such as the aorta-gonad mesonephros (AGM) due to very low HSC activity content early in development. Intra-bone marrow transplantation (IBMT) has emerged as a superior administration route over intravenous (IV) transplantation for assessing the reconstituting ability of human HSCs in the xenotransplant setting since it bypasses the requirement for homing to the BM. In this study, we compared the ability of IBMT and IV administration of embryonic day 11.5 AGM-derived cells to reconstitute the hematopoietic system of myeloablated recipients. IBMT resulted in higher levels of AGM HSC long-term multilineage engraftment in the peripheral blood, BM, spleen, and thymus of primary and secondary recipients, and in limiting dilution experiments. The administration route did not skew the multilineage contribution pattern, but IBMT conferred higher Lineage(-)Sca-1(+)c-kit(+) long-term engraftment, in line with the superior IBMT reconstitution. Therefore, IBMT represents a superior administration route to detect HSC activity from developmentally early sources with limited HSC activity content, such as the AGM.