In sexually reproducing plants, the meiocyte-producing archesporal cell lineage is maintained at the diploid state to consolidate the formation of haploid gametes. In search of molecular factors that regulate this ploidy consistency, we isolated an Arabidopsis thaliana mutant, called enlarged tetrad2 (et2), which produces tetraploid meiocytes through the stochastic occurrence of premeiotic endomitosis. Endomitotic polyploidization events were induced by alterations in cell wall formation, and similar cytokinetic defects were sporadically observed in other tissues, including cotyledons and leaves. ET2 encodes GLUCAN SYNTHASE-LIKE8 (GSL8), a callose synthase that mediates the deposition of callose at developing cell plates, root hairs, and plasmodesmata. Unlike other gsl8 mutants, in which defects in cell plate formation are seedling lethal, cytokinetic defects in et2 predominantly occur in flowers and have little effect on vegetative growth and development. Similarly, mutations in STEROL METHYLTRANSFERASE2 (SMT2), a major sterol biosynthesis enzyme, also lead to weak cytokinetic defects, primarily in the flowers. In addition, SMT2 allelic mutants also generate tetraploid meiocytes through the ectopic induction of premeiotic endomitosis. These observations demonstrate that appropriate callose and sterol biosynthesis are required for maintaining the ploidy level of the premeiotic germ lineage and that subtle defects in cytokinesis may lead to diploid gametes and polyploid offspring.