Trisomy 21 results in Down syndrome (DS) and causes phenotypes that may result from alterations of developmental processes. The Ts65Dn mouse is the most widely used genetic and phenotypic model for DS. We used over 1,500 offspring from Ts65Dn and two nontrisomic genetically similar control strains to investigate the influence of trisomy on developmental alterations and number of offspring. For the first time, we demonstrate gross developmental attenuation of Ts65Dn trisomic offspring at embryonic day (E) 9.5 and E13.5 and show that the major determinant of the developmental changes is segmental trisomy of the embryo and not the trisomic maternal uterine environment. Maternal alleles of nontrisomic genes linked to Pde6b may also influence the development of Ts65Dn offspring. Both developmental attenuation and the contribution of trisomic and nontrisomic genes are important components in the genesis of DS phenotypes. Developmental Dynamics 239:1645–1653, 2010. © 2010 Wiley-Liss, Inc.