The characteristics of cellulose nanofibers (CNFs) depend on many factors such as the raw material, type and intensity of the pre-treatment, and type and severity of the mechanical defibrillation process. The relationship among factors is complex but crucial in determining the final, fit-for-use CNF properties. This study aims to find the relationship between the CNF properties morphology, aspect ratio, nanofibrillation yield, transmittance and cationic demand, and the production process using bleached Aspen thermomechanical pulp as the raw material. Five different types of pretreatments were carried out and five different defibrillation intensities of high-pressure homogenization were evaluated. Pretreatments were: PFI refining at 20,000 revolutions, enzymatic hydrolysis with 80 and 240 g of enzyme per ton of dry pulp and TEMPO (2,2,6,6-tetramethylpiperidine-1-oxyl)–mediated oxidation with 5 and 15 mmol of NaClO per gram of dry pulp. From the twenty-five different procedures evaluated, results show that both the pretreatment and the severity of the high-pressure homogenization determined both the fibrillation yield and the CNF morphology. Moreover, the main properties of CNFs (cationic demand, yield, transmittance and aspect ratio) can be estimated from the carboxylic content of the pretreated pulp, which would facilitate the control of the CNF production and their tuning according to the production needs.