The sinoatrial node (SAN) is the primary pacemaker of the heart and controls heart rate throughout life. Failure of SAN function due to congenital disease or aging results in slowing of the heart rate and inefficient blood circulation, a condition treated by implantation of an electronic pacemaker. The ability to produce pacemaker cells in vitro could lead to an alternative, biological pacemaker therapy in which the failing SAN is replaced through cell transplantation. Here we describe a transgene-independent method for the generation of SAN-like pacemaker cells (SANLPCs) from human pluripotent stem cells by stage-specific manipulation of developmental signaling pathways. SANLPCs are identified as NKX2-5- cardiomyocytes that express markers of the SAN lineage and display typical pacemaker action potentials, ion current profiles and chronotropic responses. When transplanted into the apex of rat hearts, SANLPCs are able to pace the host tissue, demonstrating their capacity to function as a biological pacemaker.