Dictyostelium discoideum is a eukaryotic micro-organism with a unique life cycle. The amoebae live as haploid, free-living cells in the soil feeding on bacteria and dividing asexually. Under starvation conditions, the cells aggregate and undergo a process of differentiation into spores and stalk cells. We speculate that the stalk cells are sacrificed to help raise the spores above the substrate and to improve their dispersal and survival. In the case of a mix between two or more genetically different clones, a conflict may arise over which cells become spores and survive and which become stalk and die. One that differentiates more spores than its fair share in chimera is called a "cheater" and the other a "loser". Dictyostelium discoideum is a useful organism for studying the complexity of social behavior in microorganisms. Molecular tools have been developed allowing the study of genetic mechanisms that underlie this social behavior. To investigate the molecular basis of cooperation, several pools of knock-out mutants were generated using the REMI (R&barbelow;estriction E&barbelow;nzyme M&barbelow;ediated I&barbelow;ntegration) technique. To simulate evolutionary selection for cheaters, the different mutants were subjected to rounds of spore germination, growth and development in a mixed population. Only the spores were taken to the next generation. Real Time PCR confirmed that cheaters became over-represented in the evolving population because they contribute spores with a higher efficiency than the other mutants. Mutants expressing a normal phenotype were picked and isolated after 10 and 20 cycles of selection and mixed in pairwise experiments with the parental wild type. At least 35 mutants have been tested and 29 were cheaters. Analysis of the isolated genes suggested that several genetic pathways are involved in regulating or modulating the complex cooperation process in Dictyostelium discoideum. Finally we characterized one cheater mutant, called chtB, which shows apparently normal phenotype when plated clonally. The mutant is lacking in the expression of the gene chtB. In chimeras, this causes the reduction of the expression of the prespore marker cotB in the wild type strain, enabling the cheater to differentiate more spores than the parental strain.