Brown adipose tissue (BAT) is considered of metabolic significance in mammalian physiology, because it plays an important role in regulating energy balance. Alterations in this tissue have been associated with obesity and type 2 diabetes. The molecular mechanisms modulating brown adipocyte differentiation are not fully understood. Using a murine brown preadipocyte cell line, primary cultures, and 3T3-L1 cells, we analyzed the contribution of various intracellular signaling pathways to adipogenic and thermogenic programs. Sequential activation of p38MAPK and LKB1-AMPK-tuberous sclerosis complex 2 (TSC2) as well as significant attenuation of ERK1/2 and mammalian target of rapamycin (mTOR)-p70 S6 kinase 1 (p70S6K1) activation was observed through the brown differentiation process. This study demonstrates a critical role for AMPK in controlling the mTOR-p70S6K1 signaling cascade in brown but not in 3T3-L1 adipocytes. We observed that mTOR activity is essential in the first stages of differentiation. Nevertheless, subsequent inhibition of this cascade by AMPK activation is also necessary at later stages. An in vivo study showed that prolonged 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR)-induced AMPK activation increases uncoupling protein 1 expression and induces an accumulation of brown adipocytes in white adipose tissue (WAT), as revealed by immunohistology. Moreover, the induction of brown adipogenesis in areas of white fat partially correlates with the body weight reduction detected in response to treatment with AICAR. Taken together, our study reveals that differentiation of brown adipocytes employs different signaling pathways from white adipocytes, with AMPK-mTOR cross talk a central mediator of this process. Promotion of BAT development in WAT by pharmacological activation of AMPK may have potential in treating obesity by acting on energy dissipation.