Early in ovarian differentiation, female mouse germ cells develop in clusters called oocyte nests or germline cysts. After birth, mouse germ cell nests break down into individual oocytes that are surrounded by somatic pregranulosa cells to form primordial follicles. Previously, we have shown that mice treated neonatally with genistein, the primary soy phytoestrogen, have multi-oocyte follicles (MOFs), an effect apparently mediated by estrogen receptor 2 (ESR2, more commonly known as ERbeta). To determine if genistein treatment leads to MOFs by inhibiting breakdown of oocyte nests, mice were treated neonatally with genistein (50 mg/kg per day) on Days 1â5, and the differentiation of the ovary was compared with untreated controls. Mice treated with genistein had fewer single oocytes and a higher percentage of oocytes not enclosed in follicles. Oocytes from genistein-treated mice exhibited intercellular bridges at 4 days of age, long after disappearing in controls by 2 days of age. There was also an increase in the number of oocytes that survived during the nest breakdown period and fewer oocytes undergoing apoptosis on Neonatal Day 3 in genistein-treated mice as determined by poly (ADP-ribose) polymerase (PARP1) and deoxynucleotidyl transferase mediated deoxyuridine triphosphate nick end-labeling (TUNEL). These data taken together suggest that genistein exposure during development alters ovarian differentiation by inhibiting oocyte nest breakdown and attenuating oocyte cell death. Abstract Neonatal exposure to the soy phytoestrogen genistein causes disruption of ovarian differentiation, including inhibition of oocyte nest breakdown, retention of oocyte intercellular bridges, attenuation of oocyte cell death, and increased oocyte numbers.