Background: Preschool involves an array of new social experiences that may impact the development of early externalizing behavior problems over the transition to grade school. Methods: Using longitudinal data from a nationally representative sample of over 600 pairs of US twins, we tested whether the genetic and environmental influences on externalizing problems differed between children who did versus did not attend preschool. Results: At age 4, the genetic and environmental etiology of externalizing did not differ by preschool attendance. In contrast, by age 5 years (kindergarten age), the genetic and environmental etiology of externalizing significantly differed by preschool attendance. Among children who did not attend preschool, externalizing at age 5 was predominantly due to environmental influences (52% shared environment, 34% non-shared environment) rather than genetic differences (13%), whereas among children who had attended preschool, externalizing at age 5 was primarily due to genes (67%), and shared environmental influences were negligible (0%). These interactions represented the differential longitudinal persistence of genes and environments that contributed to externalizing at age 4. Sensitivity analyses ruled out confounding due to early mental ability, socioeconomic status, minority status, child age, and prior history of childcare. Conclusions: These results indicate that preschool enrollment is associated with increased genetic and decreased shared environmental influences on the development of early externalizing behavior problems. (Contains 1 table, 3 figures, and 2 footnotes.)