Cell therapy based on chimeric antigen receptor (CAR) T cells has represented a revolutionary new approach for treating tumors, especially hematological diseases. Complete remission rates (CRR) >
80%-97% and 50%-90% overall response rates (ORR) have been achieved with a treatment based on CAR-T cells in patients with malignant B-cell tumors that have relapsed or are refractory to previous treatments. Toxicity remains the major problem. Most patients treated with CAR-T cells develop high-grade cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS). However, the unprecedentedly high CRR and ORR have led to the approval of six CAR-T cell therapeutics by the Food and Drug Administration (FDA) and the European Medicines Agency (EMA), prompting researchers to improve existing products and develop new ones. By now, around 1000 clinical trials based on CAR-T cells are registered at ClinicalTrials.gov: 82% are for hematological diseases, while the remaining 16% are for solid tumors. As a result of this increased research, an enormous amount of conflicting information has been accumulated in the literature, and each group follows its manufacturing protocols and performs specific in vitro testing. This review aimed to combine and compare clinical and preclinical information, highlighting the most used protocols to provide a comprehensive overview of the in vitro world of CAR-T cells, from manufacturing to their characterization. The focus is on all steps of the CAR-T cell manufacturing process, from the collection of patient or donor blood to the enrichment of T cells, their activation with anti-CD3/CD28 beads, interleukin-2 (IL-2) or IL-7 and IL-15 (induction of a more functional memory phenotype), and their transfection (viral or non-viral methods). Automation is crucial for ensuring a standardized final product.