St John was the second son of Sir John St John (d. 1594) of Lydiard Tregoze and his wife Lucy, the daughter of Sir Walter Hungerford. Upon his father's death in 1594, most of the family estates, in Wiltshire, went to his elder brother Walter; John received the manor of Garsington, Oxfordshire, and was promised £200 when he came of age. He was first placed in the guardianship of a distant relative, John St John, 2nd Baron St John of Bletso, but Lord St John died in 1596, and guardianship then passed to John's uncle, Sir Oliver St John. Sir Oliver arranged for John's education: he matriculated at Trinity College, Oxford, on 3 April 1601 and was admitted to Lincoln's Inn in 1604. Meanwhile, John's brother Walter drowned near Castle Cornet in August 1597 while staying with Sir Thomas Leighton, Governor of Guernsey. From him, John inherited Lydiard Tregoze and the rest of the family estates in Wiltshire. His mother died in 1598, leaving him her jointure estates of Purley Park, Berkshire, and Hatfield Peverel, Essex. Sir Thomas Leighton purchased his wardship in 1602, and received permission to settle various estates on his daughter Anne, who married St John on 9 July 1604 at the age of 12. The couple had nine sons, of whom only two survived him, and four daughters, three of whom survived him. Anne died on 19 September 1628.