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(1) Background: Acute kidney injury (AKI) is a serious complication of hematopoietic stem cell transplantation (HSCT). (2) Methods: The aim was to identify the incidence, severity, and risk factors for AKI during the first 100 days after allo-HSCT; we performed a prospective observational study on 135 consecutive patients. (3) Results: The mean age was 38.3 ± 11.9 years (50.6% females), AKI developed in 93 patients (68.9%), the median time of appearance was 28 days, and the mean serum creatinine at the time of AKI was 1.8 ± 0.8 mg/dL. A total of 36 (38.7%) patients developed stage 1 AKI, 33 (35.5%) patients developed stage 2, and 24 (25.8%) patients developed stage 3; eight (8.6%) patients required temporary hemodialysis, and the mortality rate in these patients was 87.5%. Death was twice as frequent in the AKI subgroup, without statistical significance. Cyclosporine overdose (HR = 2.36, 95% CI: 1.45–3.85, p = 0.001), tacrolimus overdose (HR = 4.72, 95% CI: 2.22–10.01, p < 0.001), acute graft-versus-host disease (aGVHD) (HR = 1.96, 95% CI: 1.13–3.40, p = 0.01), and CRP level (HR = 1.009, 95% CI: 1.007–1.10, p < 0.001) were independent risk factors for AKI. Sepsis (HR = 5.37, 95% CI: 1.75–16.48, p = 0.003) and sinusoidal obstruction syndrome (HR = 5.10, 95% CI: 2.02–12.85, p = 0.001) were found as independent risk factors for AKI stage 3. (4) Conclusions: AKI occurs with high incidence and increased severity after allo-HSCT. Careful monitoring of calcineurin inhibitors and proper management of sepsis may reduce this risk.