The sexually dimorphic nucleus in the preoptic area (SDN-POA) is larger in males than in females, however the mechanism of sexual differentiation of this structure remains largely unknown, except that aromatizable androgen or estrogen during perinatal period cause the establishment of male typical nucleus. We have shown recently that enhanced green fluorescent protein (EGFP) is expressed in the SDN-POA under the control of an estrogen receptor α gene promoter 0/B (0/B-SDN). In the present study, we observed the sexual differentiation of the 0/B-SDN in vitro using the organotypic brain slice cultures, which were prepared from embryonic day 18 brains. Brain slices were cultured in the presence or absence of steroid hormones and observed at 200 hours in vitro. Estrogen or aromataizable androgen, testosterone propionate, in the culture medium masculinized the 0/B-SDN but non-aromatizable androgen, dihydrotestosterone, could not. Moreover, antagonist of estrogen receptor prevented the masculinization of 0/B-SDN even in the presence of estrogen in the culture medium. These results suggest that sexual differentiation of the 0/B-SDN could be established in vitro as well as in vivo. Next, we visualized the nucleogenesis of the 0/B-SDN in the organotypic slice culture using time-lapse imaging under the culture medium including steroid hormones or not. On the first day in vitro, no fluorescence was observed in the preoptic area of brain slices, which were prepared from embryonic day 18 brains. Outline of the 0/B-SDN appeared in the slice at 48 hours in vitro and many EGFP expressed cells aggregated prior to 100 hours in vitro. These phenomena appeared to be same, even in the absence or presence of estrogen. On the other hand, after the aggregation of EGFP cells, further diverging migration was observed in the 0/B-SDN using the culture medium including estrogen, however, 0/B-SDN cells further migrated in a converging fashion in the control medium without estrogen. These results propose the regulation of the neural migration by estrogen is important for the sexual differentiation of the SDN-POA.