Background: Preschool-aged children with acute lymphoblastic leukemia (ALL) receive long-term treatment according to the Taiwan Pediatric Oncology Group (TPOG)-ALL 2013 protocol. Severe anxiety and noncompliance ahead of frequent invasive therapies leads to an increase in health care costs. Previous studies have shown that therapeutic video games (TVGs) can decrease the anxiety experienced by children who are ill. To our knowledge, no existing TVG has been designed specifically for preschool-aged children with ALL in Taiwan., Objective: The purpose of this study was to develop a TVG using the popular Mechanics, Dynamics, and Aesthetics (MDA) framework for game design and to investigate the effect of this TVG on the reduction of therapy-related anxiety among preschool-aged children with ALL., Methods: This study used a mixed methods approach over three phases: (1) develop a TVG using the MDA framework, (2) test the reliability of the TVG among three certified children's art therapists, and (3) evaluate the reduction of therapy-related anxiety among participants after using the TVG for 6 weeks, using a two-group, stratified randomized controlled trial at a medical center in northern Taiwan. Eligible preschool-aged children with ALL were randomly assigned 1:1 into an experimental group or a control group. The two groups of subjects received the same usual care, and only the experimental group had access to and used the TVG. The children's anxiety responses were reported by their family caregivers using the face rating scale (FRS). Descriptive analyses, the Fisher exact test, the Pearson chi-square test, and the Mann-Whitney U test were used to statistically analyze the variables., Results: Six mechanics rules supported the dynamics of the TVG using four main features-character, nursery, tasks, and market-in order to complete all of the therapy-related anxiety reduction scenarios and to achieve eight aesthetics goals. The results of reliability test showed that participants found the TVG to be useful and trustworthy for preschool-aged children with ALL (Cronbach α=.98). A total of 15 participants were enrolled and randomly allocated to the experimental group (n=7) or the control group (n=8). The average number of TVG log-ins was 37.9 (SD 15.30, range 14-62) in the experimental group. The demographic data showed homogeneity across the two groups regarding age (3 to 5 years), sex (male), risk classification (standard risk), and treatment status (continuation therapy). The mean FRS score was 6.16 (SD 3.31) for the experimental group as compared to 7.45 (SD 2.71) for the control group (P=.04), which represented a significant difference between the groups at the 6-week follow-up., Conclusions: This research provides evidence that using a TVG can decrease anxiety in preschool-aged children with ALL in Taiwan. The TVG could be used to support clinical professionals before they perform invasive therapies. However, it is recommended to increase the statistical power for inference., Trial Registration: ClinicalTrials.gov NCT04199637; https://www.clinicaltrials.gov/ct2/show/NCT04199637., (©Dai-Jie Yang, Meng-Yao Lu, Chi-Wen Chen, Pei-Ching Liu, I-Ching Hou. Originally published in JMIR Serious Games (https://games.jmir.org), 22.08.2022.)