Estrogen deficiency stimulates both osteoclastic bone resorption and pre‐B lymphopoiesis, the interrelationships between which remain unknown. To investigate the involvement of an increase in the number of B220⁺ cells in accelerated osteoclastogenesis after estrogen deficiency, we first examined whether ovariectomy (OVX) increased the frequency of clonogenic osteoclast precursors in bone marrow. The results were that after OVX, the frequency of clonogenic osteoclast precursors is increased in bone marrow, suggesting that accumulated osteoclast precursors contribute to accelerated osteoclastogenesis. Further, we found that cocultures of B220⁺ cells purified from bone marrow cells and stromal ST2 cells in the presence of 1,25‐dihydroxyvitamin D₃ [1,25(OH)₂D₃] gave rise to osteoclasts that can resorb bone and express calcitonin receptors. When the frequencies of clonogenic osteoclast precursors in the purified B220⁺ and B220⁻ cell fractions were compared, it was found that the fractions gave rise to osteoclasts at similar frequencies, which rules out the possibility of cross‐contamination and suggests that the two fractions contain comparable numbers of osteoclast precursors. Furthermore, we identified cells that are positive for both tartrate‐resistant acid phosphatase (TRAP) and B220, not only in cocultures of B220⁺ and ST2 cells, but also in freshly isolated unfractionated bone cells. Therefore, it is concluded that at least a subfraction of B220⁺ cells are capable of generating osteoclasts and that the increase in the number of B220⁺ cells caused by estrogen deficiency may contribute to accelerated bone resorption by this novel osteoclastogenesis pathway.