Abstract Background Calbindin-D28 has been used as a marker for the sexually dimorphic nucleus of the preoptic area (SDN-POA). Males have a distinct cluster of calbindin-immunoreactive (ir) cells in the medial preoptic area (CALB-SDN) that is reduced or absent in females. However, it is not clear whether the sex difference is due to the absolute number of calbindin-ir cells or to cell position (that is, spread), and the cellular mechanisms underlying the sex difference are not known. We examined the number of cells in the CALB-SDN and surrounding regions of C57Bl/6 mice and used mice lacking the pro-death gene, Bax, to test the hypothesis that observed sex differences are due to cell death. Methods Experiment 1 compared the number of cells in the CALB-SDN and surrounding regions in adult males, females, and females injected with estradiol benzoate on the day of birth. In experiment 2, cell number in the CALB-SDN and adjacent regions were compared in wild-type and Bax knockout mice of both sexes. In addition, calbindin-ir cells were quantified within the principal nucleus of the bed nucleus of the stria terminalis (BNSTp), a nearby region that is larger in males due to Bax-dependent cell death. Results Males had more cells in the CALB-SDN as well as in surrounding regions than did females, and estradiol treatment of females at birth masculinized both measures. Bax deletion had no effect on cell number in the CALB-SDN or surrounding regions but increased calbindin-ir cell number in the BNSTp. Conclusions The sex difference in the CALB-SDN of mice results from an estrogen-dependent difference in cell number with no evidence found for greater spread of cells in females. Blocking Bax-dependent cell death does not prevent sex differences in calbindin-ir cell number in the BNST or CALB-SDN but increases calbindin-ir cell number in the BNSTp of both sexes.