Cells engrafted in runx1 mutants have capacity for serially repopulating. (A) Experimental schema: donor marrow was harvested from 6-month-old highly engrafted (>70% myeloid chimerism) runx1 primary transplanted fish (1° txp), and secondary transplants (2° txp) were performed into runx1-mutant embryos 2 dpf. Chimerism in secondary transplant recipients was assessed at 2 months posttransplant by flow cytometry. Tertiary transplants (3° txp) were performed with the same procedure using 6-month-old secondary recipients as donors. (B) Multilineage chimerism (myeloid [My], erythroid [Ery], lymphoid [Ly], and precursor [Pre]) in runx1-mutant recipients after secondary and tertiary transplant measured at 2 months posttransplant. The yellow line indicates 5% chimerism cutoff for engraftment. Thirty-one of 32 surviving transplanted runx1 mutants demonstrated high levels of multilineage engraftment after secondary transplantation, with 9 of 47 showing high-level engraftment after tertiary transplantation. (C) Lineage distribution of donor-derived GFP⁺ cells in individual primary and secondary transplanted donors as well as secondary and tertiary engrafted runx1-mutant recipients at 2 months posttransplant. (D) The percentage of donor-derived cells of myeloid lineage in recipients of primary, secondary, and tertiary transplantation. Secondary transplants demonstrated a significantly higher contribution from the myeloid lineage than primary recipients, but this effect was not seen in tertiary transplants. Error bars are standard deviation. Statistical analysis was performed via Mann-Whitney U test. **P < .001; ****P < .0001.