Proliferative and differentiative signals controlling the in vitro IgM response by unprimed, affinity-enriched B cells were studied using conditions under which as few as 2,000 B cells stimulated by antigen-specific, Ia-positive, allogeneically restricted, T cell-derived helper factor (Hf) or the polyclonal activator lipopolysaccharide (LPS) yielded on the average 400 antibody-forming cells (AFC) by direct plaque assay. Antigen alone induces neither B cell proliferation nor differentiation into AFC. Proliferation but not differentiation into AFC is induced when affinity-enriched B cells are cultured in the presence of Ag and Hf or LPS but in the absence of nonantigen-specific, radioresistant, accessory (A) cells. For the induction of a complete Hf- or LPS-mediated AFC response, cultures must be reconstituted with A cells or the secretory product(s) of these cells. The antigen-specific response depends strictly on the presence of the Hf specific for the relevant antigen, regardless of the cell cycle state of cooperating B cells. The differentiative signal from A cells is due, at least in part, to the presence of a Thy-1.2-bearing population of cells. In the case of the LPS-mediated, but not the Hf-mediated response. A cells can be substituted by using supernatant derived from an interleukin 2-secreting T lymphoma cell line (EL4). In the presence of histocompatible Hf and B cells, histoincompatible A cells can still cooperate in the immune response. However, the degree of allogeneic restriction between incompatible Hf and B cells is markedly increased if both B cells and A cells are incompatible with Hf.