4-Methyl-5-pentylbenzene-1,3-diol (MPBD), a product of the polyketide synthase SteelyA, is a signaling molecule that regulates Dictyostelium discoideum development. During early development, MPBD controls chemotactic cell aggregation by regulating the expression of genes in the cAMP signaling pathway; however, during culmination at late development, it induces spore maturation. In the present study, we analyzed the effects of MPBD, its derivatives, and a putative MPBD-derived metabolite on developmental defects in the MPBD-less stlA null mutant. Using structure-activity relationship studies, it was observed that in MPBD, the functional groups that were essential for induction of spore maturation were different from those essential for induction of cell aggregation. Dictyoquinone, a putative MPBD metabolite rescued the aggregation defect in stlA null mutant in early development, but not the spore maturation defect at the later stage. Our data suggest that MPBD regulates chemotactic cell aggregation and spore maturation via different mechanisms.