The current study examined the relationship between separation anxiety in adolescents after their transition to middle school, on the one hand, and differentiation of self and separation anxiety in their parents, on the other hand. The sample included 88 adolescents from northern Israel, together with their biological parents. Adolescents' separation anxiety was found to be negatively associated with maternal I-position (IP). In terms of gender differences, mothers' levels of emotional reactivity and fusion with others were higher than fathers', while fathers' levels of IP and emotional cut-off were higher than mothers'. Furthermore, parents' levels of differentiation of self were negatively correlated with their own levels of separation anxiety. The study found an absence of gender differences in separation anxiety, both for parents and adolescents. Also, parents' separation anxiety was not related to adolescents' separation anxiety. The results indicate that differentiation of self among mothers is a significant characteristic in understanding adolescents' separation anxiety. On a practical level, this study has the potential to increase the awareness of teachers, school counsellors and psychologists of those familial sources that promote and increase adolescents' separation anxiety.