Down syndrome (DS; trisomy 21) significantly impacts the human population, affecting up to six million people, and understanding the underlying mechanisms is crucial for developing effective interventions to enhance health and quality of life. Animal models, especially mouse models, have been instrumental in this area of research. Over the past two decades, Cre/loxP-mediated chromosome engineering technology has transformed DS research in mice. This technology allows for the creation of predetermined chromosomal duplications and deletions in regions syntenic to human chromosome 21. In this chapter, we discuss how this technology has led to the development of novel DS models, enabled genetic dissection of syntenic regions to establish precise genotype–phenotype relationships, and revealed the effects of an extra chromosome beyond gene dosage. These advances pave the way for a new era of discoveries in understanding this significant medical condition.