Stimulation of B cells by surface immunoglobulin (sIg) triggering, or through the mitogenic combination of phorbol ester and calcium ionophore, is accompanied by activation of STAT transcription factors. The mechanism responsible for the delayed nuclear accumulation of phosphorylated STAT3 was examined in detail, focusing on the role of B cell-derived lymphokines. sIg-induced activation of STAT3 was partially inhibited in B cells obtained from IL-6- or IL-10-deficient mice, and was partially blocked by neutralizing antibodies directed against either of these lymphokines. sIg-induced STAT3 activation was completely inhibited by combining IL-6- and IL-10-specific neutralizing antibodies, or by adding individual neutralizing antibodies to B cells obtained from lymphokine-deficient animals. In contrast, IL-10 alone appeared to account for STAT3 activation resulting from B cell stimulation with phorbol ester and calcium ionophore. In keeping with these results, soluble IL-6 and IL-10 were found in supernatant fluid obtained from stimulated B cells. This work indicates that a lymphokine pathway is responsible for STAT3 activation that occurs late after B cell stimulation, and points out differences in B cell activation that result from stimulation through the antigen receptor and through pharmacological mimicry of signaling mediators.