Background Hematopoietic stem cell transplantation (HSCT) from HLA-haploidentical related donors is increasingly considered for transplant candidates who lack a histocompatible related or unrelated donor. Since October 2015, we have been performing haploidentical PBSCT, utilizing a two-step approach first described by Grosso et al. (Biol Blood Marrow Transplant 21:646, 2015), in which the lymphoid and CD34+ cell populations are administered separately. Selective depletion of alloreactive T-cell clones is accomplished by administering high-dose cyclophosphamide (CY) after the donor lymphocyte infusion but prior to infusion of the CD34-selected peripheral blood stem cell product. Methods As of June 2019, 15 pts have undergone haploidentical PBSCT from related donors for treatment of AML (n=1), ALL (n=8), MDS (n=3), CML (n=1) JMML (n=1) and HLH (n=1). Median age at transplant was 11 years (range, 0.7-23.5 years). Twelve pts were conditioned with TBI 1200 cGy and CY 60 mg/kg IV x 2, while 3 pts received fludarabine 35 mg/m2 IV x 4, busulfan 0.8-1 mg/kg IV q6h x 16 doses, and CY 60 mg/kg IV x 2. The mother served as the donor for 8 pts, the father for 6 pts and an adult sister for 1 pt. Donor lymphocytes containing 2 × 108 CD3+ cells/kg were infused after completion of TBI or busulfan, depending on the conditioning regimen utilized, followed 66 hours later by administration of CY 60 mg/kg daily x 2. CD34+ selected peripheral blood stem cells were infused 48 hours after the second dose of CY. GvHD prophylaxis consisted of tacrolimus and mycophenolate, both begun on Day -1. In the absence of GvHD, mycophenolate was discontinued at Day +30 and tacrolimus was tapered over 1 month, beginning on Day +60. Results Median CD34+ cell dose was 7.9 × 106/kg (range, 4.1-12.3). Neutrophil and platelet (>20,000/microliter) engraftment occurred at a median of 9 days (range, 8-10 days) and 9.5 days (range, 8-20 days), respectively. All pts achieved full donor chimerism, with no secondary graft failure. Pts were discharged a median of 22 days post-transplant (range, 15-77 days). Grade II-III acute GvHD (mostly transient skin involvement) was observed in 5 pts and 1 pt developed Stage IV acute liver GvHD. Chronic skin GvHD (1 mild, 1 extensive) was observed in 2 pts. Two pts have died, one from progressive disease and one from multiorgan failure in the setting of severe acute liver GvHD. Six pts (1 JMML, 1 AML, 1 MDS, 3 ALL) have suffered disease relapse, 68-446 days post-transplant. All 3 ALL pts subsequently received CAR T-cell therapy. Eight pts remain relapse-free survivors a median of 2.4 years (range, 0.3-3.8 years) post-transplant. Conclusion Haploidentical HSCT using this two-step methodology is a safe and effective approach for pts lacking a suitably compatible donor. Relapse of disease remains the most frequent obstacle to successful treatment.