The serine/threonine kinase LKB1 is a master kinase that regulates a number of critical events such as cell transformation, polarization, development, stress response, and energy metabolism in metazoa. After multiple unsuccessful attempts of generating Dictyostelium lkb1-null cells, an RNAi-based knockdown approach proved effective. Depletion of lkb1 with a knockdown construct displayed severe reduction in prespore cell differentiation and precocious induction of prestalk cells, which were reminiscent of cells lacking GSK3. Similar to gsk3⁻ cells, lkb1 depleted cells displayed lower GSK3 activity than wild type cells during development and compromised cAMP-mediated inhibition of the DIF-1 mediated ecmB induction. In response to stress insult, the kinase activity of LKB1, but not that of GSK3, increased. Therefore, LKB1 positively functions at the upstream of GSK3 during development and responds to stress insults independently from GSK3.