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The COVID-19 pandemic brought major logistical challenges to hematopoietic stem cell transplantation, leading to the routine cryopreservation of allogeneic grafts. This study assessed the impact of ex vivo time (time from end of apheresis to infusion or cryopreservation), cell concentration, and cryopreservation on the composition and viability of 586 allogeneic peripheral blood stem cell grafts collected between April 2020 and December 2023. Grafts from unrelated donors (URD, n = 360), haploidentical related donors (HAPLO, n = 109), and matched sibling donors (SIB, n = 117) were analyzed via flow cytometry upon arrival and post-thaw. Approximately 40% of grafts were collected locally, and the remainder from international donors across Europe, the Americas, and Asia. CD34(+) hematopoietic stem cells (0.8% of graft) showed high viability on arrival (99.0%) and post-thaw (84.5%). Longer ex vivo times significantly reduced the viability of CD34(+) cells (R-2 = 0.038, P < 0.001, n = 564) and, to a greater extent, CD3(+) T cells (R-2 = 0.167, P < 0.001, n = 561). Higher cell concentrations improved CD45(+) viability (R-2 = 0.013) and correlated with better T-cell viability, including CD3(+) (R-2 = 0.020), CD4(+) (R-2 = 0.020), and CD8(+) (R-2<0.028). Interestingly, cryopreservation markedly reduced T-cell viability, with post-thaw viabilities of 65.8% for CD3(+), 60.0% for CD4(+), and 74.5% for CD8(+). Multivariable regression confirmed ex vivo time and WBC concentration as independent predictors of post-thaw CD3(+) viability. A combined variable (PM-Index) of ex vivo time and cell concentration negatively correlated with post-thaw T-cell viability (R-2 = 0.099). These findings highlight that while CD34(+) cells withstand cryopreservation, T cells are more vulnerable, stressing the need for improved graft handling protocols to ensure successful transplantation.