Background: Long-term clinical evaluation of patient outcomes can steer treatment choices and further research for cartilage repair. Using mesenchymal stromal cells (MSCs) as signaling cells instead of stem cells is a novel approach in the field. Purpose: To report the 5-year follow-up of safety, clinical efficacy, and durability after treatment of symptomatic cartilage defects in the knee with allogenic MSCs mixed with recycled autologous chondrons in first-in-human study of 1-stage cartilage repair. Study Design: Case series; Level of evidence, 4. Methods: This study is an investigator-driven study aiming at the feasibility and safety of this innovative cartilage repair procedure. Between 2013 and 2014, a total of 35 patients (mean ± SD age, 36 ± 8 years) were treated with a 1-stage cartilage repair procedure called IMPACT (Instant MSC Product Accompanying Autologous Chondron Transplantation) for a symptomatic cartilage defect on the femoral condyle or trochlear groove. Subsequent follow-up after initial publication was performed annually using online patient-reported outcome measures with a mean follow-up of 61 months (range, 56-71 months). Patient-reported outcome measures included the KOOS (Knee injury and Osteoarthritis Outcome Score), visual analog scale for pain, and EuroQol–5 Dimensions. All clinical data and serious adverse events, including additional treatment received after IMPACT, were recorded. A failure of IMPACT was defined as a chondral defect of at least 20% of the index lesion with a need for a reintervention including a surgical procedure or an intra-articular injection. Results: Using allogenic MSCs, no signs of a foreign body response or serious adverse reactions were recorded after 5 years. The majority of patients showed statistically significant and clinically relevant improvement in the KOOS and all its subscales from baseline to 60 months: overall, 57.9 ± 16.3 to 78.9 ± 17.7 (P < .001); Pain, 62.3 ± 18.9 to 79.9 ± 20.0 (P = .03); Function, 61.6 ± 16.5 to 79.4 ± 17.3 (P = .01); Activities of Daily Living, 69.0 ± 19.0 to 89.9 ± 14.9 (P < .001); Sports and Recreation, 32.3 ± 22.6 to 57.5 ± 30.0 (P = .02); and Quality of Life, 25.9 ± 12.9 to 55.8 ± 26.8 (P < .001). The visual analog scale score for pain improved significantly from baseline (45.3 ± 23.6) to 60 months (15.4 ± 13.4) (P < .001). Five cases required reintervention. Conclusion: This is the first study showing the midterm safety and efficacy of the proof of concept that allogenic MSCs augment 1-stage articular cartilage repair. The absence of serious adverse events and the clinical outcome support the longevity of this unique concept. These data support MSC-augmented chondron transplantation (IMPACT) as a safe 1-stage surgical solution that is considerably more cost-effective and a logistically advantageous alternative to conventional 2-stage cell-based therapy for articular chondral defects in the knee.